DE102005013846A1 - Identification of surface-associated antigens for tumor diagnosis and therapy - Google Patents

Abstract

In accordance with the invention, tumor-associated gene products and the nucleic acids coding therefor have been identified. The present invention relates to the therapy and diagnosis of diseases in which these tumor-associated gene products are aberrantly expressed. Furthermore, the invention relates to proteins, polypeptides and peptides which are expressed tumor-associated, and the nucleic acids coding therefor.

Description

In spite of interdisciplinary approaches and spreading classical therapies include cancer to the leading ones Causes of death. Newer therapeutic concepts aim to the patient's own immune system through the use of recombinant Tumor vaccines and other specific measures such as antibody therapy into the overall therapeutic concept. requirement for the Success of such a strategy is the detection of tumor-specific or tumor-associated antigens or epitopes by the immune system of the patient whose effector functions are interventionally enhanced should. Tumor cells differ significantly from biological their non-malignant cells of origin. These differences are through while the tumor development acquired genetic changes and cause u.a. also for the formation qualitatively or quantitatively changed molecular structures in the cancer cells. Become such tumor-associated Structures of the specific immune system of the tumor-bearing host Recognized, it is called tumor-associated antigens.

The specific recognition of tumor-associated antigens involves cellular and humoral mechanisms, which represent two units which are functionally crosslinked with one another: CD4 ⁺ and CD8 ⁺ T lymphocytes recognize processed antigens which are located on the molecules of the MHC (Major Histocompatibility Complex = Histocompatibility Complex). Antigens) classes II or I, while B lymphocytes produce circulating antibody molecules that bind directly to unprocessed antigens.

The potential clinical-therapeutic significance of tumor-associated Antigens arises from the fact that the detection of antigens on neoplastic cells by the immune system for initiation of cytotoxic effector mechanisms and in the presence of Helper T cells can cause the elimination of cancer cells (Pardoll, Nat. Med. 4: 525-31, 1998). Accordingly, it is a central objective tumor immunology, to define these structures molecularly. The The molecular nature of these antigens has long remained enigmatic. Only when appropriate cloning techniques were developed, it was possible by analyzing the target structures of cytotoxic T lymphocytes (CTL) (van der Bruggen et al., Science 254: 1643-7, 1991) or with circulating autoantibodies (Sahin et al., Curr. Opin., Immunol., 9: 709-16, 1997) as probe cDNA expression libraries tumors to screen systematically for tumor-associated antigens. For this purpose, cDNA expression libraries were prepared from fresh tumor tissue and recombinantly expressed in suitable systems as proteins. Immune effectors isolated from patients, namely CTL clones with tumor-specific Lysis patterns, or circulating autoantibodies were used to determine the to clone respective antigens.

By these approaches In recent years, a variety of antigens in various neoplasms been defined. Of great Interest is the class of Cancer / Testis antigens (CTA). CTA and her coding Genes (Cancer / Testis genes or CTG) are characterized by their characteristic expression pattern defined (Türeci et al., Mol. Med. Today 3: 342-9, 1997). They do not find themselves in Normal tissues except for testis or germ cells, however, are in one Series of human malignancies, and not tumor type-specific, but with different frequency in tumor entities of very different origin (Chen & Old, Cancer J. Sci. Am. 5: 16-7, 1999). Also serum reactivities against CTA are not found in healthy controls, but only in tumor patients. In particular, due to their tissue distribution this antigen class is of particular value for immunotherapeutic projects and is being tested in ongoing clinical patient trials (Marchand et al., Int J. Cancer 80: 219-30, 1999, Knuth et al. Cancer Chemother. Pharmacol. 46: S46-51, 2000).

Indeed use the classical methods of antigen identification outlined above Immune effectors (circulating autoantibodies or CTL clones) from patients with usually advanced cancer as probes. Out a number of data indicate that tumors e.g. for tolerization and anergization of T cells can and especially in the course of the disease those specificities lost to the immunoeffector repertoire, which is an effective Immune detection could cause. Ongoing patient studies have not yet secured evidence for one actual Effect of previously discovered and used tumor-associated antigens result. Accordingly, it can not be ruled out that spontaneous immune responses evoking proteins are the wrong target structures.

It The object of the present invention was target structures for a diagnosis and to provide therapy for cancers.

These The object is achieved by the Subject of the claims solved.

In accordance with the present invention, a strategy has been developed for identifying and providing tumor-associated expressed antigens and those coding for them Tracked nucleic acids. This strategy is based on the evaluation of human protein and nucleic acid databases with regard to potential, cell-surface-accessible, cancer-specific antigens. The definition of the necessary filter criteria together with a high-throughput methodology for the analysis of as many proteins as possible form the central component of the invention. By data mining, as complete a list as possible of all known genes is set up, which are examined in accordance with the basic principle of gene to mRNA to protein following the presence of one or more transmembrane domains out. This is followed by a homology search, a classification of the hits in tissue-specific groups (including tumor tissue) and a review of the real existence of the mRNA. Finally, the proteins thus identified are evaluated for their aberrant activation in tumors, for example by expression analyzes and protein-chemical methods.

Data Mining is a known method of identifying tumor-associated Genes. In the conventional However, strategies usually become transcriptomics of normal tissue banks electronically subtracted from tumor tissue banks on the assumption that the remaining Genes are tumor-specific (Schmitt et al., Nucleic Acids Res. 27: 4251-60, 1999; Vasmatzis et al., Proc. Natl. Acad. Sci. USA. 95: 300-4, 1998; Scheurle et al., Cancer Res. 60: 4037-43, 2000).

The inventive concept, which has proved to be much more successful, however, is based on Data mining for the electronic extraction of all genes necessary for on the cell surface accessible, cancer-specific Encoding antigens and then using them for ectopic expression in tumors.

Consequently In one aspect, the invention relates to a strategy for identification of differentially expressed in tumors genes. This combined Datamining of public Sequence banks ("in silico ") with following evaluating laboratory-experimental ("wet bench") investigations.

A Combined strategy based on different bioinformatics Scripts enabled According to the invention, the identification of genes responsible for on the cell surface accessible, cancer-specific Encode antigens. The identification and provision of this Tumor-associated genes and the gene products encoded thereby took place according to the invention independent of an immunogenic effect.

The identified according to the invention Tumor-associated antigens have an amino acid sequence derived from a nucleic acid which is selected from the group consisting of (a) one Nucleic acid, which comprises a nucleic acid sequence, selected from the group is, consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 269, 271, 273, 275, 277, 279, 309 of the Sequence Listing, a part or derivative thereof (b) of a nucleic acid which is under stringent Conditions with the nucleic acid hybridized under (a), (c) a nucleic acid which, with respect to the nucleic acid under (a) or (b) is degenerate, and (d) a nucleic acid which to the nucleic acid under (a), (b) or (c) is complementary. In a preferred embodiment has an inventively identified Tumor-associated Antigen an amino acid sequence on top of a nucleic acid which is selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 269, 271, 273, 275, 277, 279, 309 of the Sequence Listing. In another preferred embodiment includes an inventively identified Tumor-associated antigen is an amino acid sequence selected from the group selected is, consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 270, 272, 274, 276, 278, 280 to 308, 310 of the Sequence Listing, a part or derivative thereof.

The The present invention relates generally to the use of tumor-associated identified according to the invention Antigens or parts or derivatives thereof, coding therefrom nucleic acids or of nucleic acids, which are directed against the coding nucleic acids, or of antibodies those against the tumor-associated identified according to the invention Antigens or parts or derivatives thereof are directed for therapy and diagnosis. This use can be individual, but also combinations of several of these antigens, functional fragments, nucleic acids, antibodies, etc. in one embodiment also in combination with other tumor-associated genes and antigens for diagnosis, Therapy and follow-up.

The property of the inventively identified tumor-associated antigens that they or located on the cell surface qualifies them as suitable targets or agents for therapy and diagnosis. Particularly suitable for this purpose is part of the tumor-associated antigens identified according to the invention, which corresponds to or is comprised by the non-transmembrane component, in particular the extracellular component of the antigens. Thus, according to the invention, part of the tumor-associated antigens identified according to the invention which corresponds to or is comprised of the non-transmembrane portion of the antigens or a corresponding portion of the nucleic acids coding for the antigens identified according to the invention is preferred for therapy or diagnosis. Similarly, it is preferred to use antibodies directed against a portion of the tumor-associated antigens identified according to the present invention which corresponds to or is comprised by the non-transmembrane portion of the antigens.

preferred Diseases for a therapy and / or diagnosis are those in which a selective Expression or abnormal expression of one or more of the identified according to the invention Tumor-associated antigens is present.

The The invention also relates to gene products, i. Nucleic acids and Proteins or peptides that are expressed tumor-associated and through changed Splice (Splice variants) known genes or by altered Translation using alternative open reading frames arise. In this aspect, the invention relates to nucleic acids comprising a nucleic acid sequence selected from the group consisting of the sequences according to SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 269, 271, 273, 275, 277, 279, 309 of the sequence listing. Furthermore, the invention relates to In this aspect, proteins or peptides that have an amino acid sequence selected from the group consisting of the sequences according to SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 270, 272, 274, 276, 278, 280 to 308, 310 of the Sequence Listing. The splice variants according to the invention are according to the invention as targets for diagnostics and therapy of tumor diseases.

• – die Nutzung variabler Transkriptionsinitiationsstellen
• – die Nutzung zusätzlicher Exons
• – vollständiges oder unvollständiges Ausspleißen von einzelnen oder mehreren Exons,
• – über Mutation veränderte Spleißregulatorsequenzen (Deletion bzw. Schaffung neuer Donor/Akzeptorsequenzen),
• – die unvollständige Elimination von Intronsequenzen.

For the emergence of splice variants a variety of mechanisms can be causal, for example

• - the use of variable transcription initiation sites
• - the use of additional exons
• Complete or incomplete splicing of single or multiple exons,
• Mutation-modified splice regulatory sequences (deletion or creation of new donor / acceptor sequences),
• - the incomplete elimination of intron sequences.

The changed Splice a gene leads to an altered one Transcript sequence (splice variant). Will a splice variant in the field of their changed Sequence translated results in a modified protein, which is from the original one clearly distinguishable in structure and function. In tumor-associated splice variants can tumor-associated transcripts and tumor-associated proteins / antigens arise. these can as molecular markers for the detection of tumor cells as well used for the therapeutic targeting of tumors. The detection of tumor cells e.g. in the blood, serum, bone marrow, sputum, bronchial lavage, body secretions and tissue biopsies may be performed according to the invention e.g. after extraction of nucleic acids by PCR amplification with splice variant-specific oligonucleotides respectively.

For detection, all sequence-dependent detection systems are suitable according to the invention. In addition to PCR, these are eg gene chip / microarray systems, Northern blot, RNAse protection assays (RDA) and others. All detection systems have in common that the detection is based on a specific hybridization with at least one splice variant-specific nucleic acid sequence. However, the detection of tumor cells can also be carried out according to the invention by antibodies that recognize a coded by the splice variant specific epitope. For the production of the antibodies peptides for immunization can be used that are specific for this splice variant. In particular, in this aspect, the invention relates to peptides comprising a sequence selected from SEQ ID NOs: 268, 270, 272, 274, 276, 278, 280 and 281 of the Sequence Listing and to specific antibodies directed against them. The detection of tumor cells can also be carried out by antibodies that recognize tumor-specific altered Glykosylierungsvarianten. For the generation of such antibodies, peptide regions can be used which differ in tumor cells and healthy cells due to glycosylation. In particular, in this aspect, the invention relates to peptides comprising a sequence selected from SEQ ID NOs: 268, 270, 272, 274, 276, 278, 280 and 281 of the Sequence Listing and to specific antibodies directed against them. Endogenous deglycosylation of N-linked sugar residues results in Aspa ragin transformed into aspartic acid. According to the invention, therefore, the proteins described herein may be tumor-specific sequence-modified and thus have different biochemical and antibody binding properties. Particularly suitable for the immunization are the amino acids which have distinct epitope differences to the variant (s) of the gene product, which are preferably formed in healthy cells. The detection of tumor cells with antibodies can be done on a sample isolated from the patient or as imaging with intravenously applied antibodies.

Next the diagnostic usability provide splice variants, the new or altered epitopes have attractive targets for the immunotherapy. The epitopes according to the invention can be used for targeting used by therapeutically effective monoclonal antibodies or T lymphocytes become. In passive immunotherapy this antibody or T lymphocytes adoptively transferred, the splice variant-specific epitopes detect. The generation of antibodies can be like other antigens also using standard technologies (immunization of animals, Panning strategies for the isolation of recombinant antibodies) under use of polypeptides containing these epitopes. alternative can to immunize nucleic acids be used for Oligo- or polypeptides encode these epitopes. Different techniques for In vitro or in vivo generation of epitope-specific T lymphocytes are known and detailed described e.g. (Kessler JH, et al., 2001, Sahin et al., 1997) and are also based on the use of oligo- or polypeptides, the splice variant-specific Epitopes include, or nucleic acids that code for them. Oligo or Polypeptides that contain the splice variant-specific Include epitopes, or nucleic acids encoding these polypeptides, are also for the use as pharmaceutically active substances in the active Immunotherapy (vaccination, vaccine therapy) usable.

The aberrant expression of genes in tumor cells may be due to altered methylation patterns of their Promoters are based (De Smet C et al., Mol. Cell Biol. 24: 4781-90, 2004; De Smet C., et al., Mol. Cell Biol. 19: 7327-35, 1999; De Smet C. et al. Proc. Natl. Acad. Sci. USA. 93: 7149-53, 1996). These methylation differences can as an indirect marker for changed in the tumor State of the corresponding gene can be used. Accordingly, can the increase or decrease of base methylations in the promoter region for diagnostic Purposes are used.

In In another aspect, the invention also relates post-translationally modified protein domains such as glycosylations or myristilations. This kind from modifications can lead to a differential recognition pattern an antigen by e.g. lead an antibody and various, under circumstances disease-associated conditions detect. Especially through the use of antibodies can this differentiation an antigen used both diagnostically and therapeutically become. For Tumor cells is published that tumor-associated cellular degeneration to changed post-translational Modifications lead Can (Durand & Seta, Clin. Chem. 46: 795-8052000; Granovsky et al., Nat. Med. 6: 306-312, 2000). In particular, glycosylation patterns are greatly altered on tumor cells. According to the invention, these special epitopes diagnostic tumor cells of non-carcinogenic Distinguish cells. Should be a posttranslational modifiable Epitope glycosylated in normal, non-degenerated cells and in Tumor cells deglycosylated, this situation allows the development of a Tumor-specific therapeutic antibody in the context of the invention.

The Analysis of protein modifications can be done in Western Blot. Especially glycosylations, which are usually a size of several kDa have lead to a larger total mass of the target protein, which can be separated in SDS-PAGE. To the Detection of specific O- and N-glycosidic bonds Protein lysates prior to denaturation by SDS with O- or N-glycosylases incubated (according to the manufacturer's instructions, for example PNgase, Endoglycosidase F, endoglycosidase H, Roche Diagnostics). Subsequently, a Western blot. When reducing the size of a target protein can so after incubation with a glycosidase a specific glycosylation demonstrated and in this way also the tumor specificity of a Modification are analyzed. Of particular interest are protein regions, which is differentially glycosylated in tumor cells and healthy cells are. However, such glycosylation differences have so far been few Cell surface proteins (e.g., Muc1).

In one aspect, the invention relates to a pharmaceutical composition comprising an agent which recognizes the tumor-associated antigen identified according to the invention and is preferably selective for cells having expression or abnormal expression of a tumor-associated antigen identified according to the invention. In certain embodiments, the agent may cause induction of cell death, reduction of cell growth, damage to the cell membrane, or secretion of cytokines, and preferably has antitumor activity. In one embodiment, the agent is an antisense nucleic acid that selectively hybridizes to the nucleic acid encoding the tumor-associated antigen. In another embodiment, the agent is an anti body that selectively binds to the tumor-associated antigen, in particular a complement-activated antibody or toxin-conjugated antibody that selectively binds to the tumor-associated antigen. In another embodiment, the agent comprises a plurality of agents, each of which selectively recognizes various tumor-associated antigens, wherein at least one of the tumor-associated antigens is a tumor-associated antigen identified according to the invention. The recognition need not be directly associated with an inhibition of the activity or expression of the antigen. In this aspect of the invention, the antigen selectively restricted to tumors preferably serves as a label for recruiting effector mechanisms at that specific site. In a preferred embodiment, the agent is a cytotoxic T lymphocyte which recognizes the antigen on an HLA molecule and lyses the cell so labeled. In another embodiment, the agent is an antibody that selectively binds to the tumor-associated antigen, thus recruiting natural or artificial effector mechanisms to that cell. In another embodiment, the agent is a helper T lymphocyte that enhances effector functions of other cells that specifically recognize that antigen.

In In one aspect, the invention relates to a pharmaceutical composition, comprising an agent expressing the expression or activity of a identified according to the invention Tumor-associated Antigens inhibits. In a preferred embodiment, the agent is an antisense nucleic acid which selectively with the nucleic acid hybridized for the tumor-associated Encoded antigen. In a further embodiment, the agent is a Antibody, which binds selectively to the tumor-associated antigen. In another embodiment For example, the agent comprises several agents, each selectively expressing or activity inhibit various tumor-associated antigens, at least one of the tumor-associated Antigens identified according to the invention Tumor-associated antigen.

The activity an inventively identified Tumor-associated antigen can be any activity of a Be protein or peptide. Thus, the therapeutic and diagnostic methods also on inhibition or reduction of this activity or on a test of this activity are targeting.

Of Furthermore, the invention relates to a pharmaceutical composition, which comprises an agent which, when administered, selectively releases the Amount of complexes between an HLA molecule and a peptide epitope identified according to the invention Tumor-associated Antigen increased. The means comprises in one embodiment one or more constituents selected from the group consisting from (i) the tumor-associated antigen or a part thereof, (ii) a nucleic acid, the for the Tumor-associated antigen or a part thereof encoded, (iii) one Host cell containing the tumor-associated antigen or part of it and (iv) isolated complexes between peptide epitopes from the tumor-associated antigen and an MHC molecule. In a embodiment The agent comprises several agents, each of which selectively increases the amount Complexes between MHC molecules and Peptide epitopes of various tumor-associated antigens increase, wherein at least one of the tumor-associated antigens identified according to the invention Tumor-associated antigen.

Of Furthermore, the invention relates to a pharmaceutical composition, comprising one or more ingredients selected from the group consisting of (i) a tumor-associated identified according to the invention Antigen or a part thereof, (ii) a nucleic acid which for a identified according to the invention Tumor-associated antigen or a part thereof encoded, (iii) an antibody, which is identified to an inventively Tumor-associated antigen or a part thereof binds, (iv) one Antisense nucleic acid, which is specifically associated with a nucleic acid which is identified according to the invention Tumor-associated Antigen encodes, hybridizes, (v) a host cell which is an inventively identified Tumor-associated antigen or a part thereof expressed, and (vi) isolated complexes between one identified according to the invention Tumor-associated antigen or a part thereof and an HLA molecule.

A Nucleic acid, the for an inventively identified Tumor-associated antigen or part thereof may be encoded in the pharmaceutical composition is present in an expression vector and functionally linked to a promoter.

A in a pharmaceutical according to the invention Composition contained host cell may be the tumor-associated To secrete antigen or part of it, to express it on the surface or in addition an HLA molecule express that is associated with the tumor Antigen or part of it binds. In one embodiment the host cell expresses the HLA molecule endogenously. In another embodiment the host cell expresses the HLA molecule and / or the tumor-associated Antigen or part of it recombinant. Preferably, the host cell not proliferative. In a preferred embodiment, the host cell is an antigen-presenting Cell, in particular a dendritic cell, a monocyte or a Macrophage.

An antibody contained in a pharmaceutical composition of the invention may be a monoclonal antibody. In further embodiments, the antibody is a chimeric or humanized antibody, a fragment of a natural antibody, or a synthetic antibody that can be produced by combinatorial techniques. The antibody may be coupled to a therapeutically or diagnostically useful agent.

A in a pharmaceutical according to the invention Composition containing antisense nucleic acid may have a sequence of 6-50, in particular 10-30, 15-30 or 20-30 contiguous nucleotides from the Nucleic acid, the for identified according to the invention Encoded tumor-associated antigen.

In further embodiments binds by a pharmaceutical composition according to the invention either directly or provided by the expression of a nucleic acid Tumor-associated antigen or part of it on MHC molecules on the surface of Cells, wherein the binding preferably causes a cytolytic reaction and / or a cytokine secretion induced.

A inventive pharmaceutical composition can be a pharmaceutically acceptable carrier and / or an adjuvant. The adjuvant may consist of saponin, GM-CSF, CpG nucleotides, RNA, a cytokine or a chemokine. A pharmaceutical according to the invention Composition is preferably for the treatment of a disease used by selective or abnormal expression Expression of a tumor-associated antigen distinguishes. In a preferred embodiment the disease is cancer.

Of Furthermore, the invention relates to methods for treatment, diagnosis or monitoring, i.e. Determination of regression, course and / or outbreak, a disorder characterized by expression or abnormal expression one or more tumor-associated antigens.

In an embodiment the treatment comprises the administration of a pharmaceutical according to the invention Composition.

The inventive diagnostic method and / or Procedure for monitoring generally concern the use of means for detection and / or detection Determination or monitoring of the Amount of (i) a nucleic acid, the for the tumor-associated antigen encodes, or a part thereof and / or (ii) the tumor-associated antigen or a part thereof, and / or (iii) an antibody against the tumor-associated antigen or a part thereof and / or (iv) cytotoxic or helper T lymphocytes responsible for the tumor-associated antigen or part of it, in one of a patient isolated biological sample.

In In one aspect, the invention relates to a method for diagnosing a Disease resulting from expression or abnormal expression an inventively identified Tumor-associated antigen distinguishes. The method includes detection (i) a nucleic acid, the for the tumor-associated antigen encodes, or a part thereof and / or (ii) the detection of the tumor-associated antigen or part thereof, and / or (iii) the detection of an antibody to the tumor-associated Antigen or a part thereof and / or (iv) the detection of cytotoxic or helper T lymphocytes responsible for the tumor-associated antigen or a part thereof are specific in a biological sample isolated from a patient. In particular embodiments the detection comprises (i) the contacting of the biological sample with an agent specific to the nucleic acid responsible for the tumor-associated antigen or the part thereof, to the tumor-associated antigen or the Part of it, to the antibody or to cytotoxic or helper T lymphocytes responsible for the tumor Antigen or parts thereof, binds and (ii) the detection the complex formation between the agent and the nucleic acid or the Part of it, the tumor-associated antigen or part thereof, the antibody or the cytotoxic or helper T lymphocytes. In one embodiment The disease is characterized by expression or abnormal Expression of several different tumor-associated antigens and the detection comprises detection of multiple nucleic acids that for the several different tumor-associated Antigens encode, or parts of, the proof of several various tumor-associated antigens or parts thereof, the Detection of several antibodies, which belongs to the several different tumor-associated antigens or bind to parts of it, or the detection of several cytotoxic or helper T lymphocytes responsible for the several different tumor-associated antigens specific are. In a further embodiment is the isolated biological sample from the patient with a compared to comparable normal biological sample.

The diagnostic methods according to the invention can also use altered methylation patterns of the promoter region of the respective tumor-associated gene product. Detection of such methylation patterns can be accomplished using PCR-based methods, restriction enzymes or sequencing. A suitable test can be constructed as follows: (1) extraction of DNA from tissue pro (2) treatment of the DNA with bisulfite-containing reagents (eg as described in Clark SJ et al., Nucleic Acids Res. 22 (15): 2990-7, 1994), (3 ) Amplification of DNA by PCR, and (4) analysis by determination of the amount of sequence-specific amplification products (eg by quantitative PCR, hybridization methods such as microarray method).

The Diagnostic methods according to the invention can also a use of inventively identified Involve tumor-associated antigens as prognostic markers, for metastatization e.g. by testing the migration behavior of cells and therefore a worsening disease course too predict, which, among other things, enables the planning of a more aggressive therapy.

In In another aspect, the invention relates to a method for determining the regression, the course or the onset of a disease that characterized by the expression or abnormal expression of an inventively identified Tumor-associated antigen, comprising monitoring a sample from a patient having the disease or is suspected of contracting the disease in relation to one or several parameters selected from the group consisting of (i) the amount of nucleic acid, the for encodes the tumor-associated antigen, or part of it, (ii) the amount of tumor-associated antigen or part thereof, (iii) the amount of antibodies, which bind to the tumor-associated antigen or a part thereof, and (iv) the amount of cytolytic T cells or helper T cells, the for a complex between the tumor-associated antigen or a Part of it and a MHC molecule are specific. Preferably, the method comprises the determination of the parameter or parameters at a first time in a first Sample and at a second time in another sample, wherein by comparing the two samples the course of the disease is determined. In certain embodiments, it is distinguished the disease by the expression or abnormal expression of several different tumor-associated antigens and monitoring includes a surveillance (i) the amount of several nucleic acids, the for code the several different tumor-associated antigens, or parts thereof and / or (ii) the amount of several different tumor-associated Antigens or parts thereof and / or (iii) the amount of several Antibody, which belongs to the several different tumor-associated antigens or bind to parts thereof, and / or (iv) the amount of several cytolytic T cells or helper T cells responsible for complexes between the several various tumor-associated antigens or parts thereof and MHC molecules specific are.

One Detection of a nucleic acid or part thereof or a provision or monitoring the amount of a nucleic acid or part thereof can according to the invention with a polynucleotide probe which hybridizes specifically with the nucleic acid or the part thereof, or may be by selective amplification of the nucleic acid or of the part thereof. In one embodiment, the polynucleotide probe comprises a sequence of 6-50, especially 10-30, 15-30 or 20-30 contiguous Nucleotides from the nucleic acid.

In certain embodiments the diagnostic method according to the invention there is a selective amplification of the promoter region or a Part of it a nucleic acid, the for an inventively identified Tumor-associated antigen encoded and in the form of genomic DNA present, after treatment with a bisulfite-containing reagent. The nucleic acid is preferably before treatment with the bisulfite-containing Reagent isolated from a sample of a patient to be examined. The oligonucleotides used in such amplification preferably have a sequence similar to that with the bisulfite-containing Reagent treated nucleic acid bind, preferably completely complementary are. Preferably, the oligonucleotides are a different one Adjusted degree of methylation of the nucleic acid and condition differentiable amplification products.

One Detection of a tumor-associated antigen or a part thereof or a determination or monitoring the amount of a tumor-associated antigen or part thereof can according to the invention with a antibody done specifically to the tumor-associated antigen or part binds from it.

In certain embodiments is the tumor-associated antigen or part to be detected thereof in a complex with an MHC molecule, in particular an HLA molecule.

One Detection of an antibody or the determination or monitoring the amount of antibodies can according to the invention with a Protein or peptide that binds specifically to the antibody.

Detection of cytolytic T cells or helper T cells or the determination of the amount of cytolytic T cells or helper T cells specific for complexes between an antigen or a part thereof and MHC molecules, can be carried out according to the invention with a cell containing the complex between the antigen or the part thereof and a MHC moles presented.

The for one Proof or for a determination or monitoring used polynucleotide probe, antibody, protein or peptide or the cells are preferably detectably labeled. In particular embodiments the detectable marker is a radioactive marker or an enzyme marker. The detection of T-lymphocytes can additionally by proof of their Proliferation, its cytokine production, as well as its cytotoxic Activity, by the specific stimulation with the complex of MHC and Tumor-associated antigen or parts thereof is triggered. The detection of T-lymphocytes may also be by a recombinant MHC molecule or also a complex of several MHC molecules, which correspond to the respective one immunogenic fragment from one or more of the tumor-associated Antigens are loaded, and by contacting the specific T cell receptor done, creating specific T-lymphocytes can be identified.

In In another aspect, the invention relates to a method of treatment, Diagnosis or monitoring a disorder characterized by expression or abnormal expression an inventively identified Tumor-associated antigen, comprising the administration an antibody, which binds to the tumor-associated antigen or a part thereof and coupled with a therapeutic or diagnostic agent or substance is. The antibody can be a monoclonal antibody be. In further embodiments is the antibody a chimera or humanized antibody or a fragment of a natural one Antibody.

The The invention also relates to a method for treating a patient with a disorder characterized by expression or abnormal Expression of an inventively identified Tumor-associated antigen comprising (i) the removal a sample with immunoreactive cells from the patient, (ii) the Contacting the sample with a host cell containing the tumor-associated antigen or part of it expressed under conditions that produce cytolytic T cells against the tumor-associated antigen or favor a part of it, and (iii) introducing the cytolytic T cells into the patient in an amount that is suitable to lyse cells containing the tumor-associated antigen or a part express it. The invention also relates to cloning of the T-cell receptor of cytolytic T-cells against the tumor-associated Antigen. This can be transferred to other T cells, the with it the desired specificity and introduced into the patient as in (iii) can.

In an embodiment the host cell expresses an endogenous HLA molecule. In another embodiment the host cell expresses an HLA molecule and / or the tumor-associated antigen or the part thereof recombinant. Preferably, the host cell not proliferative. In a preferred embodiment, the host cell is an antigen-presenting Cell, in particular a dendritic cell, a monocyte or a Macrophage.

In In another aspect, the invention relates to a method of treatment of a patient with a disease that is manifested by expression or abnormal expression of a tumor-associated Antigen (s) comprising (i) the identification of a tumor-associated one identified according to the invention Antigen-encoding nucleic acid, which is expressed by cells associated with the disease (ii) the transfection of a host cell with the nucleic acid or part thereof; (iii) culturing the transfected host cell for expression the nucleic acid (this is not obligatory when reaching a high transfection rate) and (iv) introducing the host cells or an extract thereof into the patient in an amount that is appropriate to the immune response against the cells of the patient associated with the disease are to increase. The method may further include the identification of an MHC molecule, the presenting the tumor-associated antigen or a part thereof, wherein the host cell expresses the identified MHC molecule and presenting the tumor-associated antigen or part thereof. The immune response may be a B-cell response or a T-cell response include. Furthermore, a T cell reaction can stop production cytolytic T cells and / or helper T cells comprising specific for the host cells are the tumor-associated antigen or a Present part of it or specific to Cells of the patient are the tumor-associated antigen or express a part of it.

The Invention also relates to a method for treating a disease, which are identified by the expression or abnormal expression of an inventively identified Tumor-associated Antigen, comprising (i) the identification of cells from the patient, the abnormal amounts of the tumor-associated antigen (ii) the isolation of a sample of the cells, (iii) the Cultivating the cells; and (iv) introducing the cells into the cells Patients in an amount that is appropriate to an immune response to trigger the cells.

Preferably, the host cells used according to the invention are not proliferative or are not made proliferative. A disease characterized by the expression or abnormal expression of a tumor-associated antigen, is especially cancer.

Of Furthermore, the present invention relates to a nucleic acid, the selected from the group is composed of (a) a nucleic acid containing a nucleic acid sequence which is selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 269, 271, 273, 275, 277, 279, 309 of the Sequence Listing, a part or derivative thereof, (b) a nucleic acid, under stringent conditions with the nucleic acid under (a) hybridizes, (c) a nucleic acid which, with respect to the nucleic acid, under (a) or (b) is degenerate, and (d) a nucleic acid which to the nucleic acid under (a), (b) or (c) is complementary. Furthermore it concerns the invention a nucleic acid, the for a Protein or polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 270, 272, 274, 276, 278, 280 to 308, 310 the sequence listing, a part or derivative thereof.

In In another aspect, the invention relates to promoter sequences of Nucleic acids according to the invention. These can functional with another gene, preferably in an expression vector and thus the selective expression of this gene in appropriate cells.

In In another aspect, the invention relates to a recombinant nucleic acid molecule, in particular DNA or RNA molecule, which comprises a nucleic acid according to the invention.

The The invention also relates to host cells comprising a nucleic acid according to the invention or a recombinant nucleic acid molecule having a Nucleic acid according to the invention comprises, contain.

The The host cell may further comprise a nucleic acid encoding an HLA molecule. In one embodiment the host cell expresses the HLA molecule endogenously. In another embodiment the host cell expresses the HLA molecule and / or the nucleic acid according to the invention or a Part of it recombinant. Preferably, the host cell is not proliferative. In a preferred embodiment the host cell is an antigen-presenting cell, in particular a dendritic cell, a monocyte or a macrophage.

In a further embodiment The invention relates to oligonucleotides which are identified with an inventively identified nucleic acid hybridize and used as genetic probes or as "antisense" molecules can be. Nucleic acid molecules in the Form of oligonucleotide primers or competent samples with an inventively identified nucleic acid or parts thereof can be used to detect nucleic acids which are identified according to the invention nucleic acid are homologous. PCR amplification, Southern and Northern hybridization can to Find homologous nucleic acids be used. Hybridization may be lower, better under medium and best under highly stringent conditions. The term "stringent Conditions " according to the invention conditions allow specific hybridization between polynucleotides.

In In another aspect, the invention relates to a protein, polypeptide or peptide encoded by a nucleic acid derived from the Group selected is composed of (a) a nucleic acid containing a nucleic acid sequence consisting of the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 269, 271, 273, 275, 277, 279, 309 of the Sequence Listing a part or derivative thereof, (b) a nucleic acid, under stringent conditions with the nucleic acid under (a) hybridizes, (c) a nucleic acid which, with respect to the nucleic acid, under (a) or (b) is degenerate, and (d) a nucleic acid which to the nucleic acid under (a), (b) or (c) is complementary. In a preferred embodiment For example, the invention relates to a protein or polypeptide having an amino acid sequence includes, selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 270, 272, 274, 276, 278, 280 to 308, 310 the sequence listing, a part or derivative thereof.

In a further aspect, the invention relates to an immunogenic fragment of a tumor-associated antigen identified according to the invention. The fragment preferably binds to a human HLA receptor or human An tikörper. A fragment according to the invention preferably comprises a sequence of at least 6, in particular at least 8, at least 10, at least 12, at least 15, at least 20, at least 30 or at least 50 amino acids.

In In this aspect, the invention relates in particular to a peptide which a sequence selected from the group consisting of SEQ ID NO: 282-308, a part or derivative thereof or comprises.

In In another aspect, the invention relates to a means that an inventively identified Tumor-associated antigen or to a part thereof binds. In a preferred embodiment the agent is an antibody. In further embodiments is the antibody a chimera, a humanized or combinatorial technique antibody or a fragment of an antibody. Furthermore, the invention relates to an antibody which selectively binds to a Complex of (i) a tumor-associated antigen identified according to the invention or a part thereof and (ii) an MHC molecule to which the invention identified Tumor-associated antigen or the part thereof binds, wherein the antibodies does not bind to (i) or (ii) alone. An antibody according to the invention can a monoclonal antibody be. In further embodiments is the antibody a chimera or humanized antibody or a fragment of a natural one Antibody.

Of Furthermore, the invention relates to a conjugate between a composition according to the invention, that to an inventively identified Tumor-associated antigen or binds to a part thereof, or one antibodies of the invention and a therapeutic or diagnostic agent or substance. In an embodiment the therapeutic or diagnostic agent is a toxin.

In In another aspect, the invention relates to a kit for detection the expression or abnormal expression of an inventively identified Tumor-associated antigen comprising means for detecting (i) the Nucleic acid, the for the tumor-associated antigen encodes, or a part thereof, (ii) the tumor-associated antigen or a part thereof, (iii) of antibodies which bind to the tumor-associated antigen or a part thereof, and / or (iv) T cells responsible for a complex between the Tumor-associated antigen or a part thereof and an MHC molecule are specific. In one embodiment For example, the means for detecting the nucleic acid or portion thereof are nucleic acid molecules for the selective one Amplification of the nucleic acid, in particular a sequence of 6-50, especially 10-30, 15-30 or 20-30 contiguous Nucleotides from the nucleic acid include.

detailed Description of the invention

According to the invention Genes that are selectively expressed or aberrant in tumor cells expressed and represent tumor-associated antigens.

According to the invention these are Genes and / or their gene products and / or their derivatives and / or parts preferred target structures for therapeutic approaches. Conceptually, you can the therapeutic approaches on an inhibition of activity of the selectively expressed tumor-associated gene product. This is useful if the aberrant or selective Expression is functionally of tumor pathogenetic importance and their ligation with a selective damage to the corresponding cells accompanied. Other therapeutic concepts consider tumor-associated Antigens as markers, the effector mechanisms with cell damaging Recruit potential selectively to tumor cells. Here is the function of the target molecule itself and its role in tumorigenesis completely irrelevant.

With "derivative" of a nucleic acid according to the invention that single or multiple nucleotide substitutions, deletions and / or additions in the nucleic acid. Furthermore includes the term "derivative" is also a chemical Derivatization of a nucleic acid at a nucleotide base, at the sugar or at the phosphate of a nucleotide. Of the The term "derivative" also includes nucleic acids that contain non-naturally occurring nucleotides and nucleotide analogs.

A nucleic acid is preferred according to the invention deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). nucleic acids include genomic according to the invention DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. A nucleic acid can according to the invention as a single-stranded or double and linear or covalently circular closed molecule.

The described according to the invention nucleic acids are preferably isolated. The term "isolated nucleic acid" means according to the invention that the nucleic acid (i) was amplified in vitro, for example by polymerase chain reaction (PCR), (ii) was produced recombinantly by cloning, (iii) purified was, for example, by cleavage and gel electrophoretic separation, or (iv) has been synthesized, for example by chemical synthesis. An isolated nucleic acid is a nucleic acid that for one Manipulation by recombinant DNA techniques is available.

One nucleic acid is then another Nucleic acid "complementary" if the two sequences hybridize with one another and can form a stable duplex molecule, the hybridization preferably taking place under conditions allowing specific hybridization between polynucleotides (stringent conditions) Stringent conditions are described, for example, in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., Eds., 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, FM Ausubel et al., Eds., John Wiley & Sons, Inc., New York, and refer, for example, to hybridization at 65 ° C in hybridization buffer (3.5x SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5mM NaH ₂ PO ₄ (pH 7), 0.5% SDS, 2 mM EDTA.) SSC is a solution of 0.15 M sodium chloride and sodium citrate, pH 7, respectively. After hybridization, the membrane to which DNA has been transferred becomes, for example, 2 × SDS 0.5 × SSC / 0.1 at temperatures up to 68 ° C - SSC at room temperature and then in 0.1.

Have complementary nucleic acids at least according to the invention 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98% or at least 99% identity the nucleotides.

Nucleic acids that for tumor-associated Encode antigens alone or according to the invention in combination with other nucleic acids, in particular heterologous nucleic acids, available. In preferred embodiments is a nucleic acid functionally in conjunction with expression control sequences or regulatory sequences that are homologous with respect to the nucleic acid or heterologous. A coding sequence and a regulatory sequence are then "functionally" linked together, if they are covalently linked such that expression or transcription of the coding sequence under control or under the influence of the regulatory sequence. if the coding sequence are translated into a functional protein should, leads in a functional connection of a regulatory sequence with the coding sequence an induction of the regulatory sequence to transcribe the coding sequence without causing it a frame shift in the coding sequence or to an inability the coding sequence is translated into the desired protein or peptide to become.

Of the Term "expression control sequence" or "regulatory Sequence "includes according to the invention promoters, Enhancers and other controls that control the expression of a gene Taxes. In certain embodiments of the invention, the expression control sequences are adjustable. The exact structure of regulatory sequences can be species dependent or cell type dependent but generally includes 5'-untranscribed and 5'-untranslated Sequences involved in the initiation of transcription or translation involved are like TATA box, capping sequence, CAAT sequence and the like. In particular, 5'-non-transcribed ones Regulatory sequences a promoter region containing a promoter sequence for a transcriptional Includes control of the functionally linked gene. regulatory Sequences can also enhancer sequences or upstream include activator sequences.

To the one can So the tumor associated antigens shown here with any Expression control sequences and promoters are combined. To the but others can According to the promoters the tumor-associated gene products shown here with any other genes are combined. This allows the selective activity of this To use promoters.

Of another may be a nucleic acid according to the invention in conjunction with another nucleic acid present for a polypeptide encoding a secretion of the protein encoded by the nucleic acid or polypeptide from a host cell. Also, a nucleic acid according to the invention in combination with another nucleic acid present for encodes a polypeptide anchoring the encoded protein or polypeptide on the cell membrane of the host cell or its compartmentalization into certain organelles of this cell. Likewise connect to a nucleic acid containing a reporter gene or represents any "day".

In a preferred embodiment is a recombinant DNA molecule According to the invention, a vector, optionally with a promoter which promotes expression of a nucleic acid, e.g. a nucleic acid, the for an inventive tumor-associated Antigen encodes controls. The term "vector" is used in its most general Meaning uses and includes any intermediate vehicle for a nucleic acid, the it e.g. enable, the nucleic acid in prokaryotic and / or eukaryotic cells and, if appropriate, to integrate into a genome. Such vectors are preferably replicated and / or expressed in the cell. An intermediary Vehicle may e.g. For use in electroporation, in microprojectile bombardment, in liposomal administration, when transferred by Agrobacteria or at the insertion over DNA or RNA viruses be adjusted. Vectors include plasmids, phagemids, bacteriophages or virus genomes.

The nucleic acids, the for an inventively identified Tumor-associated antigen encoding can be used for transfection of host cells be used. With nucleic acids is meant both recombinant DNA as well as RNA. recombinant RNA can be produced by in vitro transcription from a DNA template become. It can further stabilize before application by Sequences, capping and polyadenylation are modified.

Of the Term "host cell" refers to any cell according to the invention, which can be transformed with an exogenous nucleic acid or transfectable. The term "host cells" according to the invention comprises prokaryotic (e.g., E. coli) or eukaryotic (e.g., dendritic cells, B cells, CHO cells, COS cells, K562 cells, yeast cells and insect cells). Particularly preferred are mammalian cells like cells of human, mouse, hamster, pig, goat and primate. The cells can be derived from a variety of tissue types and include primary Cells and cell lines. Specific examples include keratinocytes, peripheral blood leukocytes, bone marrow stem cells and embryonic Stem cells. In further embodiments the host cell is an antigen-presenting cell, in particular a dendritic cell, a monocyte or macrophage. A nucleic acid can present in the host cell in a single or multiple copies and is in one embodiment expressed in the host cell.

Of the Term "expression" according to the invention in its used most general meaning and includes the production of RNA or RNA and protein. It also includes partial expression of nucleic acids. Furthermore, the expression can be transient or stable. Preferred expression systems in mammalian cells include pcDNA3.l and pRc / CMV (Invitrogen, Carlsbad, CA) containing a selectable Markers contain a gene that has resistance to G418 confers (and thus a selection of stably transfected cell lines ) Allows and the cytomegalovirus (CMV) enhancer-promoter sequences.

In the cases of the invention in which an HLA molecule is a tumor-associated antigen or part of it presented, For example, an expression vector may also comprise a nucleic acid sequence encoding the HLA molecule. The nucleic acid sequence, the for the HLA molecule encoded, may be on the same expression vector as the nucleic acid, the for the tumor-associated Antigen or part of it encoded, or both nucleic acids can be present on different Expression vectors are present. In the latter case, the both expression vectors are cotransfected into a cell. If a host cell neither the tumor-associated antigen or the Part of it still the HLA molecule Both are used for expressing encoding nucleic acids either on the same expression vector or on different Expression vectors are transfected into the cell. If the cell already expressing the HLA molecule, only the nucleic acid sequence, the for the tumor-associated antigen or part thereof is encoded into the Cell transfected.

According to the invention are kits for amplifying a nucleic acid encoding a tumor-associated antigen. Such Kits include, for example, a pair of amplification primers, to the nucleic acid hybridize for the tumor-associated antigen encodes. The primers preferably comprise a sequence of 6-50, especially 10-30, 15-30 or 20-30 contiguous nucleotides from the nucleic acid and are non-overlapping, to avoid the formation of primer dimers. One of the primers will hybridize to a strand of nucleic acid that is associated with the tumor Antigen encodes and the other primer attaches to the complementary strand hybridize in an arrangement involving amplification of the nucleic acid, the for the Tumor-associated antigen encoded, allowed.

"Antisense" molecules or "antisense" nucleic acids can be used for Regulation, in particular the reduction of expression of a nucleic acid used become. The term "antisense molecule" or "antisense nucleic acid" refers to an oligonucleotide according to the invention, that is an oligoribonucleotide, oligodeoxyribonucleotide, modified Oligoribonucleotide or modified oligodeoxyribonucleotide and that under physiological conditions to DNA that has a specific Gene, or mRNA of this gene hybridizes, thereby transcribing this gene Gene and / or translation of this mRNA is inhibited. An "antisense molecule" according to the invention also includes a Construct a nucleic acid or part of it in reverse orientation with respect to its natural Contains promoter. One Antisense transcript of a nucleic acid or a part thereof can be a duplex molecule with the course occurring mRNA, which specifies the enzyme, enter and so on Accumulation of or translation of the mRNA into the active enzyme prevent. One more way is the use of ribozymes to inactivate a nucleic acid. preferred have antisense oligonucleotides according to the invention a sequence of 6-50, especially 10-30, 15-30 or 20-30 contiguous Nucleotides from the target nucleic acid are and are preferred Completely to the target nucleic acid or a part of it complementary.

In preferred embodiments, the antisense oligonucleotide hybridizes to an N-terminal or 5'-upstream site, such as a translation initiation, transcriptional initiation or promoter site. In further embodiments, the antisense oligonucleotide hybridizes to a 3 'untranslated region or mRNA splice site.

In an embodiment there is an oligonucleotide according to the invention from ribonucleotides, deoxyribonucleotides or a combination from that. Here are the 5 'end of a nucleotide and the 3'-end another nucleotide by a phosphodiester bond with each other connected. These oligonucleotides can in conventional Be synthesized or produced recombinantly.

In preferred embodiments is an oligonucleotide according to the invention a "modified" oligonucleotide. In this case, the oligonucleotide, for example, its stability or therapeutic Increase effectiveness, be modified in a variety of ways without his Ability, to bind to its target, impaired becomes. The term "modified Oligonucleotide "means an oligonucleotide according to the invention, in which (i) at least two of its nucleotides are linked by a synthetic internucleoside bond (i.e., an internucleoside bond lacking a phosphodiester bond is) linked together and / or (ii) a chemical group is covalent with the oligonucleotide which does not normally occur with nucleic acids. Preferred synthetic internucleoside bonds are phosphorothioates, Alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, Carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters and peptides.

Of the Term "modified Oligonucleotide " also oligonucleotides with a covalently modified base and / or Sugar. "Modified Oligonucleotides " For example, oligonucleotides with sugar residues, the covalently organic groups are bound with a low molecular weight, the no hydroxyl group at the 3'-position and no phosphate group the 5'-position are. Modified oligonucleotides can, for example, a 2'-O-alkylated ribose or another sugar instead of ribose such as arabinose.

The described according to the invention Proteins and polypeptides are preferably isolated. The terms "isolated Protein "or" isolated Polypeptide "mean that the protein or polypeptide is separated from its natural environment. An isolated protein or polypeptide may be present in a substantially have been cleaned up. The term "substantially purified" means that the protein or polypeptide is substantially free of other substances, with which it is present in nature or in vivo.

Such Proteins and polypeptides serve, for example, for the production of antibodies and are in an immunological or diagnostic assay or can be used as therapeutics. Proteins described in accordance with the invention and Polypeptides can be out biological samples such as tissue or cell homogenates and can also recombinant in a variety of pro- or eukaryotic expression systems are expressed.

"Derivatives" of a protein or Polypeptide or an amino acid sequence For the purposes of this invention, amino acid insertion variants include amino acid deletion variants and / or amino acid substitution variants.

• 1. kleine aliphatische, nicht-polare oder leicht-polare Reste: Ala, Ser, Thr (Pro, Gly)
• 2. negativ geladene Reste und ihre Amide: Asn, Asp, Glu, Gln
• 3. positiv geladene Reste: His, Arg, Lys
• 4. große aliphatische, nicht-polare Reste: Met, Leu, Ile, Val (Cys)
• 5. große aromatische Reste: Phe, Tyr, Trp.

Amino acid insertion variants include amino- and / or carboxy-terminal fusions, as well as insertions of single or multiple amino acids in a particular amino acid sequence. In amino acid sequence variants with an insertion, one or more amino acid residues are introduced into a predetermined site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence. Amino acid substitution variants are characterized in that at least one residue in the sequence is removed and another residue is inserted in its place. Preferably, the modifications are at positions in the amino acid sequence that are not conserved between homologous proteins or polypeptides. Preferably, amino acids are replaced by others having similar properties, such as hydrophobicity, hydrophilicity, electronegativity, side chain volume, and the like (conservative substitution). For example, conservative substitutions refer to the replacement of one amino acid by another, both amino acids being listed in the same group below:

• 1. small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr (Pro, Gly)
• 2. negatively charged residues and their amides: Asn, Asp, Glu, Gln
• 3. positively charged residues: His, Arg, Lys
• 4. large aliphatic, non-polar residues: Met, Leu, Ile, Val (Cys)
• 5. large aromatic residues: Phe, Tyr, Trp.

Three Residues are due to their special role in protein architecture in Brackets set. Gly is the only remainder without a side chain and thus gives the chain flexibility. Pro has an unusual one Geometry that severely restricts the chain. Cys can form a disulfide bridge.

The above-described amino acid Vari Antennas can be readily prepared by known peptide synthesis techniques such as Solid Phase Synthesis (Merrifield, 1964) and the like, or by recombinant DNA manipulation Techniques to introduce substitution mutations at predetermined sites into DNA that are known or partially known The manipulation of DNA sequences for the production of proteins with substitutions, insertions or deletions is described in detail, for example, in Sambrook et al. (1989).

"Derivatives" of proteins or Polypeptides according to the invention also comprise single or multiple Substitutions, deletions and / or additions of any molecules that associated with the enzyme, such as carbohydrates, lipids and / or Proteins or polypeptides. Furthermore, the term "derivative" extends to all functional ones chemical equivalents the proteins or polypeptides.

One Part or fragment of a tumor-associated antigen according to the invention has a functional Property of the polypeptide from which it is derived. Such functional properties include the interaction with antibodies that Interaction with other polypeptides or proteins that are selective Binding of nucleic acids and an enzymatic activity. An important feature is the ability to complex with To enter HLA and optionally to produce an immune response. These Immune response may be due to stimulation of cytotoxic or helper T cells based. Preferably, a part or fragment according to the invention comprises a Tumor-associated antigen a sequence of at least 6, in particular at least 8, at least 10, at least 12, at least 15, at least 20, at least 30 or at least 50 consecutive amino acids the tumor-associated antigen. A part or fragment of a tumor-associated Antigen is preferably a part of the tumor-associated antigen, the non-transmembrane portion, especially the extracellular portion of the antigen corresponds or is included.

One Part or fragment of a nucleic acid responsible for a tumor-associated antigen encoded according to the invention relates to the part the nucleic acid, at least for the tumor-associated Antigen and / or for a portion or fragment of the tumor-associated antigen such as encoded as defined above. Preferably, a part or fragment a nucleic acid, the for encodes a tumor-associated antigen, that part which belongs to the open reading frame, in particular as indicated in the Sequence Listing.

The Isolation and identification of genes responsible for tumor-associated antigens encode enabled also the diagnosis of a disease that is manifested by the expression characterized by one or more tumor-associated antigens. These methods include the determination of one or more nucleic acids that for a Encode tumor-associated antigen, and / or the determination of the encoded Tumor-associated antigens and / or peptides derived therefrom. A determination of the nucleic acid can in conventional Manner, including by polymerase chain reaction or hybridization with a labeled Probe. A determination of tumor-associated antigens or thereof Derived peptides can be detected by screening patient antisera with respect to recognition of the antigen and / or the peptides. It can also be done by screening the patient for T cells specificity for the appropriate tumor-associated antigen done.

The present invention enables also the isolation of proteins associated with tumor-associated here described Bind antigens, including antibodies and cellular Binding partner of the tumor-associated antigens.

According to the invention also in certain embodiments "dominant negative "polypeptides provided derived from tumor-associated antigens are. A dominant negative polypeptide is an inactive variant a protein that interacts with the cellular machinery active protein from its interaction with the cellular machinery repressed or competes with the active protein, whereby the effect of the active protein is reduced. For example, a dominant Negative receptor that binds ligand, but no signal produced in response to the binding of the ligand, the biological Reduce the effect of the ligand. Similarly, a dominant negative catalytically-inactive kinase normally associated with target proteins however, the target proteins do not phosphorylate, phosphorylation of the target proteins in response to a cellular signal. In similar Way, a dominant negative transcription factor attached to a promoter site in the control region of a gene, but transcription of the gene does not increase, the Effect of a normal transcription factor on the occupation of promoter binding sites without increasing transcription.

The Result of Expression of a Dominant Negative Polypeptide in a cell is a reduction in the function of active proteins. The skilled person can, for example, dominant negative variants of a protein by conventional Mutagenesis method and evaluation of the dominant negative effect of the variant polypeptide.

According to the invention are also substances such as polypeptides that bind to tumor-associated antigens. Such binders can e.g. in screening assays for a detection of tumor-associated antigens and complexes of Tumor-associated antigens with their binding partners and in a Purification of tumor-associated antigens and complexes thereof find use with their binding partners. Such substances can also for one Inhibition of activity Tumor-associated antigens, for example by binding to such Find antigens use.

According to the invention therefore binders such as e.g. Antibodies or antibody fragments, the ability have to selectively bind to tumor-associated antigens. Antibodies include polyclonal and monoclonal antibodies, in conventional Be made way.

Such antibody can Recognize proteins in their native and / or denatured state (Anderson et al., J. Immunol. 143: 1899-1904, 1989; Gardsvoll, J. Immunol. Methods 234: 107-116, 2000; Kayyem et al., Eur. J. Biochem. 208: 1-8, 1992; Spiller et al., J. Immunol. Methods 224: 51-60, 1999).

antisera the specific antibodies which bind specifically to the target protein may be different Standard methods are produced; see. for example, "Monoclonal Antibodies: A Practical Approach "by Philip Shepherd, Christopher Dean ISBN 0-19-963722-9, "Antibodies: A Laboratory Manual "by Ed Harlow, David Lane ISBN: 0879693142 and "Using Antibodies: A Laboratory Manual:" Portable Protocol NO "by Edward Harlow, David Lane, Ed. Harlow ISBN: 0879695447. And so is possible, affine and specific antibodies to generate the complex membrane proteins in their native form (Azorsa et al., J. Immunol., Methods 229: 35-48, 1999; Anderson et al., J. Immunol. 143: 1899-1904, 1989; Gardsvoll, J. Immunol. Methods. 234: 107-116, 2000). This is mainly for the production of antibodies from Meaning that should be used therapeutically, but also for many diagnostic applications. This can be done with both the entire protein, with extracellular partial sequences, as well as with cells that have the target molecule in physiologically folded Express form, be immunized.

monoclonal antibody are traditionally made with the help of the Hybridoma technology (Technical Details: see "Monoclonal Antibodies: A Practical Approach "by Philip Shepherd, Christopher Dean ISBN 0-19-963722-9; "Antibodies: A Laboratory Manual "by Ed Harlow, David Lane ISBN: 0879693142, "Using Antibodies: A Laboratory Manual: Portable Protocol NO "by Edward Harlow, David Lane, Ed Harlow ISBN: 0879695447).

It is known that only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see Clark, WR (1986), The Experimental Foundations of Modern Immunology, Wiley & Sons, Inc., New York, Roitt, I. (1991), Essential Immunology, 7th Edition, Blackwell Scientific Publications, Oxford). The pFc 'and Fc regions are, for example, effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc 'region has been enzymatically cleaved or which has been produced without the pFc' region, termed the F (ab ') ₂ fragment, carries both antigen binding sites of a complete antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved or which has been produced without the Fc region, termed a Fab fragment, carries an antigen binding site of an intact antibody molecule. Furthermore, Fab fragments consist of a covalently linked light chain of an antibody and part of the heavy chain of the antibody, designated Fd. The Fd fragments are the major determinants of antibody specificity (a single Fd fragment can be up to ten different light chains are associated without altering the specificity of the antibody) and Fd fragments retain the ability to bind to an epitope upon isolation.

Within of the antigen-binding portion of an antibody are complementarity-determining Regions (CDRs) that interact directly with the epitope of the antigen, and Scaffolding Regions (FRs), the tertiary structure of the paratops. Both in the Fd fragment of the heavy chain as well as in the light chain of IgG immunoglobulins four skeletal regions (FR1 to FR4), each by three complementarity determining Regions (CDR1 to CDR3) are separated. The CDRs and in particular the CDR3 regions and even more the CDR3 heavy chain region are mostly for the Responsible for antibody specificity.

you White, that the non-CDR regions of a mammalian antibody by similar Regions of antibodies can be replaced with the same or a different specificity, where the specificity for the Epitope of the original antibody preserved. This enabled the development of so-called "humanized" antibodies which non-human CDRs are covalently linked to human FR and / or Fc / pFc regions for the Production of a functional antibody are connected.

This is done using the so-called "SLAM" technology, which isolates B cells from whole blood and monoclonalizes the cells, then analyzes the supernatant of the isolated B cell for antibody specificity, in contrast to Hybri Dom.-technology is then the variable region of the antibody gene amplified by a single-cell PCR and cloned into a suitable vector. In this way, the recovery of monoclonal antibodies is accelerated (de Wildt et al., J. Immunol., Methods 207: 61-67, 1997).

When another example, WO 92/04381 describes the preparation and Use of mouse humanized RSV antibodies in which at least a part of mouse FR regions through FR regions of a human Origin were replaced. Such antibodies, including fragments intact antibody with an antigen-binding ability are often referred to as "chimeric" antibodies.

According to the invention, F (ab ') ₂ , Fab, Fv and Fd fragments of antibodies, chimeric antibodies, in which the Fc and / or FR and / or CDR1 and / or CDR2 and / or light Chain CDR3 regions have been replaced by homologous human or non-human sequences, chimeric F (ab ') ₂ fragment antibodies in which the FR and / or CDR1 and / or CDR2 and / or light chain CDR3 Regions were replaced by homologous human or non-human sequences, chimeric Fab fragment antibodies in which the FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions are replaced by homologous human or non-human human chimeric Fd fragment antibodies in which the FR and / or CDR1 and / or CDR2 regions have been replaced by homologous human or non-human sequences. Also included according to the invention are so-called single-chain antibodies.

Preferably is an inventively used antibody against one of the sequences according to SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 270, 272, 274, 276, 278, 280 to 308, 310 of the Sequence Listing, a part or a derivative thereof, in particular a sequence according to SEQ ID NOs: 281 to 308 of the Sequence Listing and / or can by Immunization can be obtained with these peptides.

According to the invention are also polypeptides specific to tumor-associated antigens tie. For example, you can such polypeptide binding agents by degenerate peptide libraries which are simply immobilized in solution Form or as phage display libraries. Combinatorial Libraries of peptides with one or more amino acids may also be used getting produced. Furthermore, can Libraries of peptoids and non-peptide synthetic Residues are produced.

Phage display may be particularly effective in identifying binding peptides of the invention be. In this case, for example, a phage library (by use for example, the m13, fd or lambda phage), the Inserts of a length from 4 to about 80 amino acid residues presents. Phages are then selected, carry the inserts that bind to the tumor-associated antigen. This process can be over several cycles of a backselection be repeated by phages attached to the tumor-associated antigen tie. Repeated laps lead to an accumulation of phages carrying certain sequences. It For example, an analysis of DNA sequences can be made to sequence the sequences to identify expressed polypeptides. The smallest linear Proportion of the sequence that binds to the tumor-associated antigen be determined. The "two-hybrid system" from yeast can also for the Identification of polypeptides that are associated with a tumor-associated Bind antigen. Tumor-associated described according to the invention Antigens or fragments thereof can for a Screening of peptide libraries, including phage display libraries, be used to peptide binding partner to identify and select the tumor-associated antigens. Such molecules can for example Screening Assays, Purification Protocols, for Interference with the Function of the tumor-associated antigen and for other purposes to those skilled in the art are known to be used.

For example, the antibodies and other binding molecules described above can be used to identify tissue that expresses a tumor-associated antigen. Antibodies may also be coupled to specific diagnostic agents for imaging cells and tissues that express tumor-associated antigens. They can also be coupled to therapeutically useful substances. Diagnostic agents include, but are not limited to, barium sulfate, iocetamic acid, iopanoic acid, calcium ipodate, sodium diatrizoate, meglumine diatrizoate, metrizamide, sodium tylopanoate, and radiodiagnostics, including positron emitters such as fluorine-18 and carbon-11, gamma-emitters Iodine-123, technetium-99m, iodine-131 and indium-111, nuclides for nuclear magnetic resonance such as fluorine and gadolinium. The term "therapeutically useful substance" means according to the invention any therapeutic molecule that is desirably selectively delivered to a cell that expresses one or more tumor-associated antigens, including anticancer agents, with radioactivity anticancer agents include, for example, aminoglutethimide, azathioprine, bleomycin sulfate, busulfan, carmustine, chlorambucil, cisplatin, cyclophosphamide, cyclosporin, cytarabine, dacarbazine, dactinomycin, daunorubin, doxorubicin, taxol, etoposide, fluorouracil , Interferon-α, lomustine, mercaptopurine, methotrexate, mitotane, procarbazine-HCl, thioguanine, vinblastine sulfate and vincristine sulfate. Other anticancer agents are described, for example, in Goodman and Gilman, "The Pharmacological Basis of Therapeutics", B. Auflage, 1990, McGraw-Hill, Inc., especially Chapter 52 (Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner)) Proteins such as pokeweed antiviral protein, cholera toxin, perlussistoxin, ricin, gelonin, abrin, diphtheria exotoxin, or Pseudomonas exotoxin. Toxin residues can also be high energy emitting radionuclides such as cobalt-60.

Of the Term "patient" means according to the invention human, non-human primate or another animal, in particular mammal like cow, horse, pig, sheep, goat, dog, cat or rodent like Mouse and rat. In a particularly preferred embodiment the patient is a human.

Of the The term "disease" refers to anyone according to the invention pathological condition in which tumor-associated antigens are expressed or expressed abnormally. "Abnormal expression" means according to the invention that the expression opposite the Condition changed in a healthy individual, preferably increased. An increase Expression is an increase by at least 10%, in particular at least 20%, at least 50% or at least 100%. In one embodiment the tumor-associated Antigen expressed only in tissue of a diseased individual while the Expression is repressed in a healthy individual. An example such a disease is cancer, whereby the term "cancer" according to the invention leukemias, seminomas, Melanomas, teratomas, gliomas, colon, colon, rectal, colorectal, Stomach, gastrointestinal, esophageal, cervical, nasal, ear (ENT) -, Renal, Adrenal, Thyroid, Lymph Node, Breast, prostate, uterus, Ovarian, endometrial, liver, pancreatic, dermal, cerebral and lung cancers and their metastases.

A biological sample can according to the invention a tissue and / or cellular sample be and can for a use in the various methods described herein in conventional Be obtained by tissue biopsy, including punch biopsy, and removal of blood, bronchial aspirate, urine, feces or other body fluids.

The term "immunoreactive cell" means according to the invention a cell which can mature into an immune cell (such as B-cell, helper-T-cell or cytolytic T-cell) with appropriate stimulation Immunoreactive cells include CD34 ⁺ hematopoietic stem cells, immature and mature T Cells and immature and mature B cells If the production of cytolytic or helper T cells recognizing a tumor-associated antigen is desired, the immunoreactive cell is challenged with a cell expressing a tumor-associated antigen Differentiation of T cell progenitors into a cytolytic T cell upon exposure to an antigen is similar to clonal selection of the immune system ,

Some Therapeutic procedures are based on a reaction of the immune system a patient that leads to lysis of antigen-presenting cells, such as Cancer cells presenting one or more tumor-associated antigens. For example, autologous cytotoxic T lymphocytes, the for a complex of a tumor-associated antigen and an MHC molecule are specific, administered to a patient with a cell abnormality. The production of such cytotoxic T lymphocytes in vitro is known. An example for a Methods for differentiation of T cells can be found in WO-A-96/33265. In general a sample of cells, such as blood cells, is removed from the patient and the cells are brought into contact with a cell containing the Complex presented and can cause an increase of cytotoxic T lymphocytes (e.g., dendritic cells). The target cell can be transfected Cell be like a COS cell. Present these transfected cells the wished Complex on its surface and stimulate upon contact with cytotoxic T lymphocytes their multiplication. The clonally expanded autologous cytotoxic T lymphocytes are then administered to the patient.

In another method of selecting antigen-specific cytotoxic T lymphocytes, fluorogenic tetramers of MHC class I molecule / peptide complexes are used for detection of specific clones of cytotoxic T lymphocytes (Altman et al., Science 274: 94-96 , Dunbar, et al., Curr. Biol. 8: 413-416, 1998). Soluble MHC class I molecules are folded in vitro in the presence of β ₂ -microglobulin and a peptide antigen that binds to the class I molecule. After purification of the MHC / peptide complexes, they are labeled with biotin. Tetramers are formed by mixing the biotinylated peptide-MHC complexes with labeled avidin (eg, phycoerythrin) at a 4: 1 molar ratio. Tetramers are then treated with cytotoxic T lymphocytes such as peripheral Blood or lymph nodes brought into contact. The tetramers bind to cytotoxic T lymphocytes that recognize the peptide antigen / MHC class I complex. Cells that bind to the tetramers can be sorted by fluorescence-directed cell sorting to isolate reactive cytotoxic T lymphocytes. The isolated cytotoxic T lymphocytes can then be propagated in vitro.

at a therapeutic procedure called adoptive transfer (Greenberg, J. Immunol., 136 (5): 1917, 1986; Riddle et al., Science 257: 238; 1992; Lynch et al., Eur. J. Immunol. 21: 1403-1410, 1991; Kast et al., Cell 59: 603-614, 1989), cells which are the desired Present complex (e.g., dendritic cells) with cytotoxic T lymphocytes of the patient to be treated combined, resulting in an increase of specific cytotoxic T lymphocytes leads. The increased cytotoxic T lymphocytes are then sent to a patient with a cellular abnormality administered, which is characterized by certain abnormal cells, that present the specific complex. The cytotoxic T lymphocytes then lyse the abnormal cells, making a desired therapeutic effect is achieved.

Often can be derived from the T-cell repertoire of a patient only low affinity T cells against such a specific complex multiply because the high-affine have been extinguished by tolerance development are. An alternative here is a transfer of the T cell receptor itself be. For this will be also cells that have the desired Present complex (e.g., dendritic cells) with cytotoxic T lymphocytes of Healthy combined. this leads to to an increase of high affinity specific cytotoxic T-lymphocytes, when the T lymphocytes come from a donor organism that interacts with the specific complex had previously had no contact. The high-affine T cell receptor from these increased specific T lymphocytes is cloned and can be purified by gene transfer e.g. with retroviral vectors be transduced as desired in T cells from other patients. Adoptive transfer then takes place with these genetically modified T lymphocytes (Stanislawski et al., Nat. Immunol., 2: 962-70, 2001; Kessels et al., Nat. Immunol. 2: 957-61, 2001).

The The above therapeutic aspects assume that at least Some of the patient's abnormal cells are a complex of one Presenting tumor-associated antigen and an HLA molecule. An identification such cells can be done in a conventional manner. As soon as Cells that present the complex are identified were, can she with a sample from the patient, the cytotoxic T lymphocytes contains be combined. If the cells that present the complex be lysed by the cytotoxic T lymphocytes, it can be assumed that a tumor-associated antigen is presented.

Of the Adoptive transfer is not the only form of therapy that can be used according to the invention is. Cytotoxic T lymphocytes may also be in vivo in themselves be generated in a known manner. In a procedure will not proliferative cells expressing the complex. The Cells that are used will be the ones that normally express the complex, such as irradiated tumor cells or cells, who were transfected with one or both genes for a presentation of the complex (i.e., the antigenic peptide and the presenting one HLA molecule). Different cell types can be used. Furthermore, vectors can be used carrying one or both of the genes of interest. Viral or bacterial vectors are particularly preferred. For example, nucleic acids that for a Tumor-associated Encode antigen or a part thereof, functionally with promoter and enhancer sequences linked be an expression of the tumor-associated antigen or control a fragment thereof in certain tissues or cell types. The nucleic acid can be incorporated into an expression vector. expression vectors can unmodified extrachromosomal nucleic acids, plasmids or viral Genome in which an insertion of exogenous nucleic acids is possible. nucleic acids, the for can encode a tumor-associated antigen, also in a retroviral Genome be inserted, whereby the integration of the nucleic acid in the Genome of the target tissue or the target cell is made possible. In these systems carries a microorganism such as vaccinia virus, poxvirus, herpes simplex virus, retrovirus or Adenovirus the gene of interest and "actually" infects host cells preferred form is the introduction of the tumor-associated antigen in the form of recombinant RNA. This can e.g. by liposomal Transfer or by electroporation into cells. Present the resulting cells the complex of interest and are cytotoxic by autologous T lymphocytes recognized, which then multiply.

A similar effect can be achieved by combining the tumor-associated antigen or a fragment thereof with an adjuvant to allow incorporation into antigen-presenting cells in vivo. The tumor-associated antigen or a fragment thereof may be represented as protein, as DNA (eg within a vector) or as RNA. The tumor-associated antigen is processed to give a peptide partner for the HLA molecule while a fragment there from being presented without further processing being required. The latter is especially the case when they can bind to HLA molecules. Forms of administration in which the total antigen is processed in vivo by a dendritic cell are preferred because helper T cell responses may also arise. An effective immune response requires these (Ossendorp et al., Immunol Lett., 74: 75-9, 2000; Ossendorp et al., J. Exp. Med. 187: 693-702, 1998). In general, an effective amount of the tumor-associated antigen can be administered to a patient, for example, by intradermal injection. However, the injection can also take place intranodally in a lymph node (Maloy et al., Proc Natl Acad Sci USA 98: 3299-303, 2001). It can also be used in combination with reagents that facilitate uptake into dendritic cells. Preferred tumor-associated antigens include those that react with allogeneic cancer antisera or with T cells from many cancer patients. Of particular interest, however, are also those against which no spontaneous immune responses exist. Immune responses capable of lysing tumors can be shown to be effective against them (Keogh et al., J. Immunol., 167: 787-96, 2001; Appella et al., Biomed., Pept., Nucleic Acids 1: 177-84, 1995; Wentworth et al., Mol. Immunol., 32: 603-12, 1995).

The described according to the invention Pharmaceutical compositions may also be used as vaccines for immunization be used. The terms "immunization" or "vaccination" mean according to the invention an increase or Activation of an immune response to an antigen. animal models can for a Testing an immunizing effect against cancer by use a tumor-associated antigen or a nucleic acid encoding it. For example, you can human cancer cells in a mouse for the creation of a tumor are introduced and one or more nucleic acids, the for tumor-associated Encoding antigens can be administered become. The effect on the cancer cells (for example, reduction tumor size) as a measure of effectiveness an immunization can be measured by the nucleic acid.

When Part of the composition for An immunization will be one or more tumor-associated antigens or stimulating fragments thereof with one or more adjuvants for an induction an immune response or an increase in an immune response. An adjuvant is a substance that is incorporated into the antigen or administered together with it and enhances the immune response. adjuvants can the immune response by providing an antigen reservoir (extracellular or in Macrophages), activation of macrophages and / or stimulation strengthen certain lymphocytes. Adjuvants are known and include in a non-limiting manner Monophosphoryl lipid A (MPL, SmithKline Beecham), saponins such as QS21 (SmithKline Beecham), DQS21 (SmithKline Beecham; WO 96/33739), QS7, QS17, QS18 and QS-L1 (So et al., Mol. Cells 7: 178-186, 1997), incomplete Freund's Adjuvant, complete Feud's adjuvant, vitamin E, montanide, alum, CpG nucleotides (see Krieg et al., Nature 374: 546-9, 1995) and various water-in-oil emulsions, made of biodegradable oils as squalene and / or tocopherol are produced. Preferably the peptides are administered in a mixture with DQS21 / MPL. The relationship from DQS21 to MPL typically about 1:10 to 10: 1, preferably about 1: 5 to 5: 1 and in particular about 1: 1. For an administration to humans are typically DQS21 and MPL in a vaccine formulation ranging from about 1 μg to about 100 μg present.

Other Substances that stimulate an immune response of the patient can also be administered. For example, cytokines are at a vaccination due to their regulatory properties on lymphocytes usable. Such cytokines include e.g. Interleukin-12 (IL-12), which was shown to that it's the protective one Increases effects of vaccines (see Science 268: 1432-1434, 1995), GM-CSF and IL-18.

It There are a number of compounds that enhance an immune response and which can therefore be used in a vaccination. These include co-stimulatory molecules, which are provided in the form of proteins or nucleic acids. Such co-stimulatory molecules For example, B7-1 and B7-2 (CD80 and CD86, respectively) are dendritic Cells (DC) and expressed with that expressed on the T cells Interact with the CD28 molecule. This interaction provides a co-stimulation (signal 2) for an antigen / MHC / TCR-stimulated (Signal 1) T cell ready, reducing T cell proliferation and effector function is reinforced. B7 also interacts with CTLA4 (CD152) on T cells and studies that target CTLA4 and B7 ligands show that the B7-CTLA4 interaction has anti-tumor immunity and CTL proliferation strengthen (Zheng, P. et al., Proc Natl Acad Sci., USA 95 (11): 6284-6289 (1998)).

B7 is typically not expressed on tumor cells, so they are not effective antigen presenting cells (APCs) for T cells. Induction of B7 expression would allow tumor cells to more effectively stimulate proliferation of cytotoxic T lymphocytes and effector function. Co-stimulation by a combination of B7 / IL-6 / IL-12 showed induction of the IFN gamma and Th1 cytokine profile in a T cell population, resulting in further enhanced T cell activity (Gajewski et al., J. Immunol 154: 5637-5648 (1995)).

A full Activation of cytotoxic T lymphocytes and complete effector function requires involvement of T helper cells through the interaction between the CD40 ligand on the T helper cells and the CD40 molecule derived from dendritic cells (Ridge et al., Nature 393: 474 (1998), Bennett et al., Nature 393: 478 (1998), Schonberger et al., Nature 393: 480 (1998)). The mechanism of this co-stimulating signal probably affects the increase in B7 and associated IL-6 / IL-12 production by the dendritic cells (antigen-presenting cells). The CD40-CD40L interaction thus complements the interactions of signal 1 (antigen / MHC-TCR) and the signal 2 (B7-CD28).

The Use of anti-CD40 antibodies for stimulation As expected, dendritic cells would be direct a reaction to tumor antigens strengthen normally outside the area of an inflammatory Reaction or non-professional Antigen-presenting Cells (tumor cells) presented become. In these situations, T helper and B7 co-stimulatory Signals not provided. This mechanism could be in the Related to therapies used on antigen-pulsed dendritic Cells are based, or in situations where T helper epitopes not in known TRA precursors were defined.

Provided according to the invention is also administration of nucleic acids, polypeptides or peptides. Administration of polypeptides and peptides may be per se done in a known manner. In one embodiment, the administration takes place of nucleic acids by ex vivo methods, i. by removing cells from one Patients, genetic change of the cells to incorporate a tumor-associated antigen and reintroduction the changed Cells in the patient. This generally includes the introduction a functional copy of a gene into the cells of a patient in vitro and the repatriation of the genetically modified Cells in the patient. The functional copy of the gene is below functional control of regulatory elements that have a Allow expression of the gene in the genetically modified cells. transfection and transduction methods are known in the art. Provided according to the invention is also administration of nucleic acids in vivo by use of vectors such as viruses and targeted liposomes.

In a preferred embodiment is a viral vector for the administration of a nucleic acid, the for encodes a tumor-associated antigen selected from the group consisting from adenoviruses, adeno-associated viruses, poxviruses, including vaccinia virus and attenuated poxviruses, Semliki forest virus, retroviruses, Sindbis virus and Ty virus-like Particles. Particularly preferred are adenoviruses and retroviruses. The Retroviruses are common replication-deficient (i.e., they are incapable of infectious particles to create).

Various Procedures can be used to according to the invention nucleic acids in cells in vitro or in vivo. Such methods include transfection of nucleic acid-calcium phosphate precipitates, the transfection of nucleic acids, that are associated with DEAE, transfection or infection with the above viruses carrying the nucleic acids of interest, liposome-mediated transfection and the like. In certain embodiments is a control of the nucleic acid preferred to certain cells. In such embodiments, a carrier that for administration a nucleic acid to a cell (e.g., a retrovirus or a liposome) will have a bound targeting molecule. For example, can a molecule like an antibody, the one for one Surface membrane protein is specific on the target cell, or a ligand for one Receptor on the target cell incorporated into or bound to the nucleic acid carrier become. Preferred antibodies include Antibody, which selectively bind a tumor-associated antigen. If an administration a nucleic acid desired by liposomes is, can Proteins attached to a surface membrane protein bind associated with endocytosis into the liposome formulation be installed to allow a target control and / or recording. Such Proteins include capsid proteins or fragments thereof which are suitable for a specific cell types, antibodies to proteins that internalize be, proteins that drive an intracellular site, and the like.

The therapeutic according to the invention Compositions can in pharmaceutically acceptable Preparations are administered. Such preparations may usually have pharmaceutically acceptable concentrations of salts, buffers, preservatives, carriers, supplemental immunity enhancing Substances such as adjuvants (e.g., CpG nucleotides) and cytokines and optionally contain other therapeutic agents.

The therapeutic agents of the present invention can be administered by any conventional route, including injection or infusion. The administration may be, for example, oral, intravenous, intraperitoneal, intramuscular, subcutaneous or transdermal. A Therapeutic administration of antibodies is preferably by a lung aerosol. The administration of antisense nucleic acids is preferably by slow intravenous administration.

The Compositions of the invention are administered in effective amounts. An "effective amount" refers to the amount alone or together with other doses a desired reaction or a desired one Achieved effect. In case of treatment of a certain disease or a particular condition resulting from the expression of a or more tumor-associated antigens the desired Reaction the inhibition of disease progression. This includes the slowdown the progression of the disease and in particular an interruption the progression of the disease. The desired reaction during a treatment illness or condition can also delay the delay Outbreak or prevention of the onset of the disease or of the state.

A effective amount of a composition of the invention is of the condition to be treated, the severity of the disease, the individual Parameters of the patient, including age, physiological Condition, height and weight, the Duration of treatment, type of concomitant therapy (if available), the specific route of administration and the like Depend on factors.

The pharmaceutical compositions of the invention are preferably sterile and contain an effective amount of the therapeutically effective Substance for the generation of the desired Reaction or desired Effect.

The Doses of the compositions according to the invention, which can be administered of various parameters such as the mode of administration, the condition the patient, the desired administration period, etc. depend on. In the case, that a reaction in a patient at an initial Dose is insufficient higher Cans (or effectively higher Canned by another, more localized route of administration be achieved) are used.

in the Generally be for a treatment or for a generation or increase an immune response doses of the tumor-associated antigen of 1 ng to 1 mg, preferably from 10 ng to 100 ug formulated and administered. In case of administration of nucleic acids (DNA as well as RNA) that are associated with tumor Encode antigens, desired For example, doses of 1 ng to 0.1 mg are formulated and administered.

The pharmaceutical compositions of the invention are generally in pharmaceutically acceptable amounts and in pharmaceutical acceptable Administered compositions. The term "pharmaceutically acceptable" refers to a non-toxic material, not with the effect of the active ingredient of the pharmaceutical Composition interacts. Such preparations can usually be salts, Buffering agents, preservatives, carriers and optionally others contain therapeutic agents. When used in the In medicine, the salts should be pharmaceutically acceptable. Non-pharmaceutically compatible Salts can however for the preparation of pharmaceutically acceptable salts thereof can be used and are included according to the invention. Such pharmacologically and pharmaceutically acceptable salts do not include in limiting, those made from the following acids: hydrogen chloride, Hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, Salicylic, citric, formic, malonic, succinic and the like. Pharmaceutically acceptable Salts can also as alkali metal or alkaline earth metal salts such as sodium, potassium or calcium salts.

A inventive pharmaceutical composition can be a pharmaceutically acceptable carrier include. The term "pharmaceutical compatible Carrier "according to the invention relates to a or more compatible solid or liquid fillers, diluents or capsule substances used for suitable for administration to a human. The term "carrier" refers to an organic or inorganic component, natural or synthetic Nature in which the active ingredient is combined to an application to facilitate. The components of the pharmaceutical according to the invention Compositions are usually such that no interaction occurs that has the desired pharmaceutical efficacy significantly impaired.

The pharmaceutical compositions of the invention can suitable buffering agents such as acetic acid in a salt, citric acid in one Salt, boric acid in a salt and phosphoric acid contained in a salt.

The Pharmaceutical compositions may also be suitable as appropriate Preservatives such as benzalkonium chloride, chlorobutanol, parabens and thimerosal.

The pharmaceutical compositions are usually presented in a unit dosage form and can be prepared in a manner known per se. Pharmaceutical compositions according to the invention can be used in For example, in the form of capsules, tablets, lozenges, solutions, suspensions, syrups, elixirs or as an emulsion.

compositions the for suitable for parenteral administration, usually include one sterile aqueous or non-aqueous preparation of the active ingredient, preferably isotonic with the blood of the recipient is. Compatible carriers and solvent are for example Ringer's solution and isotonic sodium chloride solution.

In addition will be usually sterile, fixed oils as a solution or suspension medium used.

The The present invention is illustrated by the following figures and Examples in detail described exclusively to serve the explanation and are not meant to be limiting. The skilled person is due the description and the examples further embodiments accessible which also comprises according to the invention are.

pictures:

1 : gPCR analysis of SEQ ID NO: 1 in normal and tumor tissues.
Quantitative expression analysis of SEQ ID NO: 1 in healthy skin tissue, testis tissue and melanomas. Logarithmic representation of relative expression (fold activation). Tumor-selective expression is evident.

2 : Conventional RT-PCR analysis of SEQ ID NO: 1 in melanomas.
RT-PCR expression analysis of SEQ ID NO: 1 (FLJ31461) in melanoma (n = 14) and melanoma cell lines (n = 4) compared to healthy skin (n = 4) and testis (n = 3).

3 : gPCR analysis of SEQ ID NO: 5 in normal and tumor tissues.
Quantitative expression analysis of SEQ ID NO: 5 in normal tissues and different tumors (pools of 3-5 individual samples each, right side). A: Logarithmic representation of relative expression (fold activation). B: Image after gel electrophoretic separation of the amplified fragments. Tumor-selective expression is evident.

4 Detailed analysis of SEQ ID NO: 5-specific expression in normal and tumor tissues.
A: Quantitative expression analysis of SEQ ID NO: 5 in various ENT, kidney and uterine tumors compared to expression in the associated normal tissues. Logarithmic representation. B: Image after gel electrophoretic separation of the amplified fragments. Tumor-selective expression is evident.

5a : Northern blot analysis with a SEQ ID NO: 5 specific sequence.
Hybridization of a DIG-labeled DNA probe prepared by PCR amplification with the primers according to SEQ ID NO: 7 and 8, with testis-specific RNA. Lane 1: 2 μg of testis-specific RNA; Lane 2: 1 μg of testis-specific RNA.

5b : Immunohistochemistry with SEQ ID NO: 5-specific antiserum.
As expected from the RT-PCR reactions, the protein according to SEQ ID NO: 5 could be detected in testicular tissue (A) as well as in renal cell carcinomas (B) using a specific antiserum.

6a , b, c: RT-PCR analysis of LOC203413 in normal and tumor tissues.
6a A: Logarithmic representation of expression (fold activation). 6a Federation 6b : Result after gel electrophoretic separation. 6c A: Linear representation of relative expression. 6c B: Result after gel electrophoretic separation of the amplificates

7 : Immunofluorescence with a LOC203413-specific antibody.
A tumor cell line was transfected with a construct encoding a fusion construct of green-fluorescence protein (GFP) and LOC203413. The areas where protein attaches are green (A). This cell was stained with the antibody (B). Overlapping (C) shows that the antibody is specific for the protein.

8a , b: gPCR analysis of LOC90625-specific expression in normal and tumor tissues.
8a : Quantitative expression analysis of LOC90625 in normal tissues (left) and tumor tissues (pools of 3-5 individual samples, right). Linear representation of relative expression (fold activation). 8b : Logarithmic representation of expression in normal and tumor tissues.

9a : Staining of LOC90625 transfectants with specific antibodies.
Cells were transfected with the specific gene as a fusion construct with eGFP (A) and stained with a specific antibody against this gene product (B). Specificity is demonstrated by the overlay (C) of the stains by immunofluorescence.

9b : Staining of LOC90625 Transfectants with Specific Antibodies in the Western Blot.
Lanes 1 & 6: 293 cells, lanes 2 & 7: 293 cells transfected with LOC90625 and tagged with GFP, Spu 3, 4, 9 & 8: 293 cells transfected with LOC90625 alone.

9c : Immunohistochemistry in tissue sections with LOC90625 antibodies.
Testis germ cells are the only normal cells that express this gene product. Tumors like the prostate cancer shown here also express.

10 : qRT-PCR analysis of FAM26A in normal and tumor tissues.
Quantitative RT-PCR expression analysis of FAM26A in ovarian, gastric, esophageal, pancreatic and hepatic carcinomas compared to the respective healthy tissue. Linear representation of relative expression (fold activation). Tumor-selective expression is evident.

11 : Characterization of FAM26A-specific antibody.
Western blot analysis of the antisera generated by immunization with the peptide of SEQ ID NO: 291 (A) and 292 (B), respectively. Extracts of CHO cells were analyzed after transfection with epitope-specific (A 1, 3, B 2, 4) or respectively epitope-nonspecific (A2, 4; B 1, 3) plasmids. The arrow indicates the specific fragments.

12 : Analysis of the FAM26A protein in tumors.
Detection of FAM26A in cervical, ovarian and pancreatic tumors using FAM26A-specific antibodies (SEQ ID NO: 292).

13 : Analysis of the FAM26A protein in cell lines.
Analysis of the FAM26A protein in cell lines using SEQ ID NO: 291-specific antibodies. Western blot analysis with preimmune serum as specificity control (lanes 1-5) and FAM26A-specific antibodies.

14 : Immunofluorescence analysis of SW480 cells with a FAM26A-specific antibody.
Constitutively expressed protein is cell membrane.

15 : Immunohistochemical analysis of FAM26A in human tissues.
A: Immunohistochemical analysis of the FAM26A protein in pancreatic carcinoma samples (40-fold magnification, dilution 1: 300) using the SEQ ID NO: 292-specific antiserum. B: germ cells of the human testicle.

16 : qRT-PCR analysis of SEMA5B-specific expression.
Quantitative expression analysis of SEMA5B in normal tissues (left) and tumor samples (pools of 3-5 individual samples, right). Linear representation of relative expression (fold activation).

17 : Detailed analysis of SEMA5B-specific expression in renal cell carcinoma samples.
Quantitative expression analysis of SEMA5B in A) renal cell carcinoma samples (T1-T12) compared to healthy kidney tissue (N; n = 3) and B) breast cancer (T1-T12) compared to healthy breast tissue (N; n = 3); Logarithmic representation of relative expression (fold activation).

18a , b, c, d: qRT-PCR analysis of GJB5-specific expression in normal and tumor tissues.

19 : Western blot analysis of a GJB5-specific antibody.
Lanes 4 & 7 are control transfected cells. Lanes 5, 6, 8 & 9 are transfected with a fusion construct of GJB5 and GFP. The specific fusion protein has the expected size of about 50 kDa.

20 : qRT-PCR analysis of KLK5-specific expression.
Quantitative expression analysis of KLK5 in healthy tissue samples (left) and tumors (pools of 3-5 individual samples, right). Linear representation of relative expression (fold activation).

21a : Evidence of the specificity of an antibody directed against KLK5.
293 cells were transfected with KLK5-encoding constructs (lane 3) and compared to controls (lanes 1 and 2).

21b : Staining of protein in immunohistochemical sections of human tumors with antibodies directed against KLK5.
The antibody is directed against an extracellular region of the protein that remains after processing the portion to be secreted. A: primary breast tumor; B: breast tumor, metastasis; C: normal mammary gland.

22 : qRT-PCR analysis of LOC352765-specific expression.
Quantitative expression analysis of LOC352765 in healthy tissue samples (left) and tumors (pools of 3-5 individual samples, right). Logarithmic representation of relative expression (fold activation).

23 : Detailed analysis of LOC352765-specific expression in different tumor types.
Quantitative expression analysis of LOC352765 in breast cancer (n = 12) compared to healthy tissue samples. Logarithmic representation of relative expression (fold activation).

24 : qRT-PCR analysis of SVCT1-specific expression.
Quantitative expression analysis of SVCT1 in healthy tissue samples (left) and tumors (pools of 3-5 individual samples, right). Logarithmic representation of relative expression (fold activation).

25 : Detailed analysis of SVCT1-specific expression in different tumor types.
Quantitative expression analysis of SVCT1 in A renal carcinomas (n = 12), B cervical tumors (n = 4) and ENT tumors (n = 5) compared to healthy tissue samples. Logarithmic representation of relative expression (fold activation).

26 : qRT-PCR analysis of LOC199953-specific expression in renal cell carcinoma and ENT tumors.
Quantitative expression analysis of LOC199953 in renal cell carcinoma (n = 12) and ENT tumors (n = 5) compared to healthy kidney and skin specific tissue samples. Linear representation of relative expression (fold activation).

27 : qRT-PCR analysis of TMEM31-specific expression.
Quantitative expression analysis of TMEM31 in healthy tissue samples (left) and tumors (pools of 3-5 individual samples, right). Logarithmic representation of relative expression (fold activation).

28 : Detailed analysis of TMEM31-specific expression in different tumor types.
Quantitative expression analysis of TMEM31 in A gastric carcinoma (n = 10) and B breast cancer (n = 12) compared to healthy tissue samples. Logarithmic representation of relative expression (fold activation).

29 : qRT-PCR analysis of FLJ25132-specific expression in ovarian tumors and prostate cancers.
Quantitative expression analysis of FLJ25132 in ovarian tumors (n = 8) and prostate carcinomas (n = 10) compared to healthy tissue samples. Linear representation of relative expression (fold activation).

30 : qRT-PCR analysis of SEQ ID NO: 57-specific expression.
Quantitative expression analysis of SEQ ID NO 57 in healthy tissue samples (left) and tumors (pools of 3-5 individual samples, right). Linear representation of relative expression (fold activation).

31 Detailed analysis of SEQ ID NO: 57-specific expression in different tumor types.
Quantitative expression analysis of SEQ ID NO: 57 in A esophageal tumors (n = 10), B liver carcinomas (n = 8), C renal carcinomas and D cervical and ENT tumors compared to healthy tissue samples. Linear (A, B, D) or logarithmic (C) representation of relative expression (fold activation).

32 : qRT-PCR analysis of LOC119395-specific expression.
Quantitative expression analysis of LOC119395 in healthy tissue samples (left) and tumors (pools of 3-5 individual samples, right). Linear representation of relative expression (fold activation).

33 : Detailed analysis of LOC119395-specific expression in different tumor types.
Quantitative expression analysis of LOC119395 in A breast tumors (n = 12), B esophageal carcinomas (n = 8) and C colon and gastric carcinomas compared to healthy tissue samples. Logarithmic representation of relative expression (fold activation).

34 : qRT-PCR analysis of LOC121838-specific expression.
A Quantitative analysis of LOC121838-specific expression in healthy tissue samples (left) and tumors (pools of 3-5 individual samples, right). Linear representation of relative expression (fold activation). B Detailed analysis of LOC121838-specific RNA in ovarian tissues, logarithmic representation.

35 : qRT-PCR analysis of LOC221103-specific expression.
Quantitative expression analysis of LOC221103 RNA in healthy tissue samples (left) and tumors (pools of 3-5 individual samples, right). Linear representation of relative expression (fold activation).

36 : Detailed qRT-PCR analysis of LOC221103-specific expression in liver samples.
Quantitative expression analysis of LOC221103 RNA in liver tumors (n = 8) and a healthy liver sample (N). Linear representation of relative expression (fold activation).

37 : qRT-PCR analysis of LOC338579-specific expression.
Quantitative expression analysis of LOC338579-specific RNA in healthy tissue samples (left) and tumors (pools of 3-5 individual samples, right). Logarithmic representation of relative expression (fold activation).

38 : qRT-PCR analysis of LOC90342-specific expression.
Quantitative expression analysis of LOC90342-specific RNA in healthy tissue samples (left) and tumors (pools of 3-5 individual samples, right). Logarithmic representation of relative expression (fold activation).

39 : qRT-PCR analysis of LRFN1-specific expression.
Quantitative expression analysis of LRFN1-specific RNA in healthy tissue samples (left) and tumors (pools of 3-5 individual samples, right). Logarithmic representation of relative expression (fold activation).

40 : qRT-PCR analysis of LOC285916-specific expression.
A Quantitative analysis of LOC285916-specific expression in healthy tissue samples (left) and tumors (pools of 3-5 individual samples, right). Linear representation of relative expression (fold activation). B Detailed analysis of LOC285916-specific RNA in kidney tissues and ENT tumors, logarithmic representation.

41 : qRT-PCR analysis of MGC71744-specific expression.
A Quantitative analysis of MGC71744-specific expression in healthy tissue samples (left) and tumors (pools of 3-5 individual samples, right). Linear representation of relative expression (fold activation). B Detailed analysis of MGC71744-specific RNA in various kidney tissues, logarithmic representation.

42 : qRT-PCR analysis of LOC342982-specific expression.
Quantitative expression analysis of LOC342982-specific RNA in healthy tissue samples (left) and tumors (pools of 3-5 individual samples, right). Logarithmic representation of relative expression (fold activation).

43 : qRT-PCR analysis of LOC343169-specific expression.
A Quantitative analysis of LOC343169-specific expression in healthy tissue samples (left) and tumors (pools of 3-5 individual samples, right). Linear representation of relative expression (fold activation). B Detailed analysis of LOC343169-specific RNA in various ovarian tissues, logarithmic representation.

44 : qRT-PCR analysis of LOC340204-specific expression.
A Quantitative analysis of LOC340204-specific expression in healthy tissue samples (left) and tumors (pools of 3-5 individual samples, right). Linear representation of relative expression (fold activation). B Gel image of selected tissue samples after gel electrophoretic separation.

45 : qRT-PCR analysis of LOC340067-specific expression.
Quantitative expression analysis of LOC340067-specific RNA in healthy tissue samples (left) and tumors (pools of 3-5 individual samples, right). Logarithmic representation of relative expression (fold activation).

46 : qRT-PCR analysis of LOC342780-specific expression.
Quantitative expression analysis of LOC342780-specific RNA in healthy tissue samples (left) and tumors (pools of 3-5 individual samples, right). Logarithmic representation of relative expression (fold activation).

47 : qRT-PCR analysis of LOC339511-specific expression.
A Quantitative analysis of LOC339511-specific expression in healthy tissue samples (left) and tumors (pools of 3-5 individual samples, right). Linear representation of relative expression (fold activation). B Detailed analysis of LOC339511-specific RNA in different liver-specific tissues, linear representation.

48 : qRT-PCR analysis of C14orf37-specific expression.
Quantitative expression analysis of C14orf37 in healthy tissue samples (left) and tumors (pools of 3-5 individual samples, right). Linear representation of relative expression (fold activation).

49 : qRT-PCR analysis of ATP1A4-specific expression.
A Quantitative expression analysis of ATP1A4 in healthy tissue samples and tumors (pools of 3-5 individual samples each). Logarithmic representation of relative expression (fold activation). B Detailed analysis of ATP1A4-specific RNA in different breast-specific tissues, logarithmic representation.

Examples:

material and methods

The Terms "in silico ", and" electronic "refer purely to the use of database-based procedures that allow also laboratory experimental procedures can be simulated.

All other terms and terms are, unless explicitly defined otherwise, used as understood by those skilled in the art. The techniques mentioned and methods are carried out in a manner known per se and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. All procedures involving the use of kits and reagents are carried out according to the manufacturer's instructions.

A. Datamining-based Strategy for the identification of tumor-associated antigens

According to the invention were public human protein and nucleic acid databases with regard to cancer specific antigens, which are based on cell surface accessible are. The definition of that necessary filter criteria together with a high throughput methodology for analysis as possible of all proteins formed the central component of this strategy.

The The starting point was formed mainly by the human genome project predicted potential genes identified in the RefSeq database (Pruitt et al., Trends Genet. Jan; 16 (1): 44-47, 2000) of the "National Center for Biotechnology Information "(NCBI) as a purely model protein (XP) or mRNA entries (XM-) are stored. In another approach, the validated protein items (NP-) or the corresponding mRNAs (NM-) of the same database analyzed in the same way. The basic principle (hypothetical) Following gene to mRNA to protein, the proteins were combined several prediction programs for protein analysis first being examined for the presence of transmembrane domains. From the human XP fraction of the "RefSeq" database have been total 19,544 entries analyzed, with 2,025 hypothetical proteins meeting the filter criteria enough. The Human NP Group delivered a total of 19,110 entries with a Proportion of 4,634 filtered proteins.

The corresponding mRNA of each of these 2,025 and 4,634 proteins, respectively subsequently a homology search in the EST database (Boguski et al., Nat. Genet. 4 (4): 332-333, 1993) of the NCBI using the "BLAST" algorithm (Altschul et al., Nucleic Acids Res. 25: 3389-3402, 1997). The filter criteria were set stringently in this search. A total of 1,270 hypothetical mRNAs achieved at least one hit in the EST database, the number of hits in individual cases was more than 1,000.

For each Some of these valid scores subsequently became the tissue-specific one Origin of the underlying cDNA library as well as the name of the Library determined. The resulting tissues were in divided into four different groups, by dispensible organs (Group 3) to absolutely vital organs (Group 0). Another group 4 formed all the samples Cancerous tissues were recovered. The distribution of hits on the five groups was recorded in a table based on the best ratio of Sum of groups 3 and 4 opposite the sum of groups 0-2 was sorted. They reached those mRNAs occupy a top spot whose EST hits exclusively cancerous tissue came from, followed by those who still in tissues to be found in the dispensable organs of group 3. Another criterion for the informative value This distribution formed the number of independent cDNA libraries from which the ESTs were derived were recorded in a separate column in the table has been.

There it is in the first approach detected transcripts and the corresponding proteins first are hypothetical constructs, were even more filter criteria involved the real existence of the mRNAs and thus also the proteins should prove. For this purpose, each mRNA was analyzed using the program "Spidey" (Wheelan et al. Genome Res. 11 (11): 1952-1957, 2001) to the predicted gene locus compared. Only those transcripts that have at least one splicing operation have, i. which were distributed to at least 2 exons were for more advanced Used analyzes.

The Sequential application of all mentioned filters led to the tumor-associated antigens according to the invention, due to a predicted transmembrane domain and the associated topology are considered to be of extracellular accessibility. The Expression profile derived from the EST data is evident in all cases a cancer-specific expression out, the most at least dispensible Organs can extend.

B. Validation strategy the tumor-associated antigens identified by in silico analysis

to Use of the targets for immunotherapeutic purposes (antibody therapy using monoclonal Antibody, Vaccination, T cell receptor-mediated therapeutic approaches; see. EP-B-0 879 282) or other targeted approaches (small compounds, siRNA etc.) in cancer therapy as well as for diagnostic issues is the validation of the inventively identified targets of central importance. The validation is carried out by expression analysis at both RNA and protein level.

1. investigation the RNA expression

The first characterization of the identified tumor antigens is carried out with the aid of RNA, which from different tissues or tissue-specific cell lines. Because the differential expression pattern of healthy tissue compared to tumor tissue is of crucial importance for the subsequent therapeutic application, the characterization of the target genes is preferably carried out with the aid of these tissue samples.

The Isolation of total RNA from native tissue samples or from tumor cell lines is done with procedures that are standard in molecular biology. For example, the isolation can be done using the RNeasy Maxi Kit (Qiagen, Cat. No. 75162) according to the instructions of the manufacturer. This isolation method relies on the use of guanidinium isothiocyanate as a chaotropic reagent. Alternatively, the isolation with acid Phenol performed become (Chomczynski & Sacchi, Anal. Biochem. 162: 156-159, 1987). After workup of the tissue Guanidinium isothiocyanate makes the RNA with acidic phenol extracted, then the RNA is precipitated with isopropanol and taken up in DEPC-treated water.

2-4 μg of the so isolated RNA are subsequently added e.g. using Superscript II (Invitrogen) according to the protocol rewritten by the manufacturer in cDNA. The priming of cDNA synthesis takes place with the help of random Hexamers (e.g., Roche Diagnostics) according to standard protocols of each Manufacturer. For quality control For example, the cDNAs are amplified with primers in 30 cycles that are specific for the are only slightly expressed p53 gene. Only p53 positive cDNA samples be for the further reaction steps used.

to detailed analysis of the targets is based on a cDNA archive, that from different normal and tumor tissues as well as tumor cell lines was isolated, an expression analysis by means of PCR or quantitative PCR (qPCR) performed. To 0.5 μl cDNA from the above approach with a DNA polymerase (e.g., 1 U HotStarTaq DNA polymerase, Qiagen) amplified analogously to the protocols of the respective manufacturer (total volume of the mixture: 25-50 μl). In addition to the polymerase contains the amplification mixture 0.3 mM dNTPs, reaction buffer (final concentration 1 x, depending from the manufacturer of the DNA polymerase) and in each case 0.3 mM of the gene-specific "sense" and "antisense" primers.

The specific primers of the target gene are, as far as possible, so selected, that they are in two different exons and thus genomic Contaminations do not lead to false positive results. at a non-quantitative endpoint PCR, the cDNA is typically 15 minutes at 95 ° C incubated to denature the DNA and to activate the hot-start enzyme. Subsequently The DNA is amplified in 35 cycles (1 min 95 ° C, 1 min primer specific Hybridization temperature (about 55-65 ° C), 1 min 72 ° C for elongation of the amplificates). 10 μl of PCR approach will follow applied on agarose gels and separated in the electric field. By dyeing with ethidium bromide, the DNA is visualized in the gels and the result of the PCR is documented by a photo.

alternative for conventional PCR, the expression analysis of a target gene also be done by quantitative real time PCR. To this analysis Meanwhile, various analysis systems are available, the most well-known the ABI PRISM Sequence Detection System (TaqMan, Applied Biosystems), the iCycler (Biorad) and the Light cycler (Roche Diagnostics). As described above, a specific PCR approach is run in the "real subjected to time "-PCR devices. By adding a DNA-intercalating dye (e.g. ethidium bromide, CybrGreen) is the newly synthesized DNA by specific light excitation (according to the dye manufacturer) made visible. Through a variety of measuring points during the amplification can track the entire process and a quantitative Determination of nucleic acid concentration of Target gene performed become. The normalization of the PCR approach is performed by measurement a "housekeeping Gene "(e.g., 18S RNA, β-actin). Alternative strategies over Fluorescence-labeled DNA probes also allow the quantitative Determination of the target gene from a specific tissue sample (see TaqMan applications of the company Applied Biosystems).

2. Cloning

The Cloning of the entire target gene, for further characterization The tumor antigen is necessary, according to common molecular biological Method (e.g., in "Current Protocols in Molecular Biology ", John Wiley & Sons Ltd., Wiley InterScience). For cloning or sequence analysis of Target gene will this first with a DNA polymerase with "proof reading function "(e.g. pfu, Roche Diagnostics). The amplificate is then with Standard procedure ligated into a cloning vector. Positive clones are identified by sequence analysis and then with Help of prediction programs and known algorithms.

3. Prediction of the protein

Lots found according to the invention Genes (especially from the XM domain of RefSeq) are gene new discoveries, for the cloned the full length gene, the open reading frame is determined and the protein sequence deduced and needs to be analyzed.

For the full-length cloning of the sequence ha We use common protocols for rapid amplification of cDNA ends, as well as screening of cDNA expression libraries with gene-specific probes (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) After assembling the fragments thus found, potential open reading frames (ORFs) have been predicted by common prediction programs, since the position of the polyA tail and polyadenylation motifs dictate the orientation of the potential gene product, leaving only a possible 6 reading frames Often, these give rise to only one sufficiently large open reading frame that can code for one protein, while the other reading frames have too many stop codons and would not code for realistic proteins, while alternative open reading frames support this consideration the Kozak criteria for optimal transcription Initiation as well as analysis of potentially resulting deduced protein sequences identifying the authentic ORF. This is further verified by generating immune sera against deduced proteins of the potential ORFs and analyzing them for recognition of a real protein in tissues and cell lines.

4. extraction of antibodies

The Characterization of the tumor-associated identified according to the invention Antigens occur, for example, through the use of antibodies. Further For example, the invention includes the diagnostic or therapeutic use of antibodies. there can antibody Recognize proteins in native and / or denatured state (Anderson et al., J. Immunol. 143: 1899-1904, 1989; Gardsvoll, J. Immunol. Methods 234: 107-116, 2000; Kayyem et al., Eur. J. Biochem. 208: 1-8, 1992; Spiller et al., J. Immunol. Methods 224: 51-60, 1999).

antisera the specific antibodies which bind specifically to the target protein may be different Standard methods are produced; see. for example, "Monoclonal Antibodies: A Practical Approach "by Philip Shepherd, Christopher Dean ISBN 0-19-963722-9, "Antibodies: A Laboratory Manual "by Ed Harlow, David Lane ISBN: 0879693142 and "Using Antibodies: A Laboratory Manual:" Portable Protocol NO "by Edward Harlow, David Lane, Ed. Harlow ISBN: 0879695447. And so is possible, affine and specific antibodies to generate the complex membrane proteins in their native form (Azorsa et al., J. Immunol., Methods 229: 35-48, 1999; Anderson et al., J. Immunol. 143: 1899-1904, 1989; Gardsvoll, J. Immunol. Methods. 234: 107-116, 2000). This is especially for the production of therapeutic antibodies important, but also has Many diagnostic applications are very important. This can both with the whole protein as well as with extracellular partial sequences be immunized.

Immunization and extraction of polyclonal antibodies

various Immunization protocols are published. One species (e.g., rabbit, mice) is immunized by a first injection of the desired target protein. By a second or third immunization within a defined Period (about 2-4 weeks after the last immunization) can be enhance the immune response of the animal to the immunogen. In turn after different defined time intervals (1st bleeding after 4 weeks, subsequently every 2-3 weeks up to 5 withdrawals) the animals are bled and gained immune serum. The immune sera thus obtained contain polyclonal Antibody, with which the target protein in the Western Blot, by the flow cytometry, Immunofluorescence or immunohistochemistry detected and characterized can be.

• (1) Im ersten Fall werden Peptide (Länge: 8-12 Aminosäuren) über in vitro Verfahren synthetisiert (durch einen kommerziellen Service möglich) und diese Peptide zur Immunisierung verwendet. In der Regel erfolgen 3 Immunisierungen (z.B. mit einer Konzentration von 5-100 μg/Immunisierung).
• (2) Alternativ kann die Immunisierung durch rekombinante Proteine erfolgen. Dazu wird die klonierte DNA des Zielgens in einen Expressionsvektor kloniert und das Zielprotein analog den Bedingungen des jeweiligen Herstellers (z.B. Roche Diagnostics, Invitrogen, Clontech, Qiagen) z.B. zellfrei in vitro, in Bakterien (z.B. E. coli), in Hefe (z.B. S. pombe), in Insektenzellen oder in Säugetierzellen synthetisiert. Dabei ist auch die Synthese des Zielproteins mit Hilfe von viralen Expressionssystemen möglich (z.B. Baculovirus, Vacciniavirus, Adenovirus). Nach Synthese in einem der Systeme wird das Zielprotein aufgereinigt. Die Aufreinigung erfolgt dabei in der Regel über chromatographische Verfahren. Dabei können auch Proteine für die Immunisierung verwendet werden, die über einen molekularen Anker als Hilfsmittel zur Reinigung verfügen (z.B. His-Tag, Qiagen; FLAG-Tag, Roche Diagnostics; GST-Fusionsproteine). Eine Vielzahl von Protokollen befinden sich z.B. in den „Current Protocols in Molecular Biology", John Wiley & Sons Ltd., Wiley InterScience. Nach Reinigung des Zielproteins erfolgt eine Immunisierung wie vorstehend beschrieben.
• (3) Falls eine Zelllinie zur Verfügung steht, die das gewünschte Protein endogen synthetisiert, kann auch diese Zelllinie direkt zur Herstellung des spezifischen Antiserums verwendet werden. Die Immunisierung erfolgt dabei in 1-3 Injektionen mit jeweils ca. 1-5 × 10⁷ Zellen.
• (4) Die Immunisierung kann auch durch Injektion von DNA (DNA-Immunisierung) erfolgen. Dazu wird das Zielgen zunächst in einen Expressionsvektor kloniert, so dass die Zielsequenz unter der Kontrolle eines starken eukaryontischen Promotors steht (z.B. CMV-Promotor). Anschließend wird DNA (z.B. 1-10 μg pro Injektion) als Immunogen mit einer „gene gun" in stark durchblutete, kapillare Bereiche eines Organismus transferiert (z.B. Maus, Kaninchen). Die transferierte DNA wird von Zellen des Tieres aufgenommen, das Zielgen wird exprimiert und das Tier entwickelt schließlich eine Immunantwort gegen das Zielprotein (Jung et al., Mol. Cells 12: 41-49, 2001; Kasinrerk et al., Hybrid Hybridomics 21: 287-293, 2002).

The animals are usually immunized by one of four well-established methods, although other methods exist. It is possible to immunize with peptides specific to the target protein, the entire protein, with extracellular partial sequences of a protein which can be identified experimentally or via prediction programs. Since the prediction programs do not always work without errors, you may also work with two domains that are separated from each other by a transmembrane domain. One of the two domains must then be extracellular, which can then be confirmed experimentally (see below). The implementation of immunization is offered commercially as a service by various service providers.

• (1) In the first case, peptides (8-12 amino acids in length) are synthesized by in vitro methods (possible through a commercial service) and these peptides are used for immunization. As a rule, 3 immunizations are carried out (eg with a concentration of 5-100 μg / immunization).
• (2) Alternatively, immunization may be by recombinant proteins. For this purpose, the cloned DNA of the target gene is cloned into an expression vector and the target protein analogously to the conditions of the respective manufacturer (eg Roche Diagnostics, Invitrogen, Clontech, Qiagen) cell-free in vitro, in bacteria (eg E. coli), in yeast (eg pombe), in insect cells or in mammalian cells. It is also the synthesis of the target protein using viral expression systems possible (eg baculovirus, vaccinia virus, adenovirus). After synthesis in one of the syste the target protein is purified. The purification is usually carried out by chromatographic methods. Proteins can also be used for immunization which have a molecular anchor as a cleaning aid (eg His-Tag, Qiagen, FLAG-Tag, Roche Diagnostics, GST fusion proteins). A variety of protocols are found, for example, in "Current Protocols in Molecular Biology," John Wiley & Sons Ltd, Wiley InterScience. After purification of the target protein, immunization is performed as described above.
• (3) If a cell line is available which endogenously synthesizes the desired protein, this cell line can also be used directly for the production of the specific antiserum. The immunization is carried out in 1-3 injections, each with about 1-5 × 10 ⁷ cells.
• (4) Immunization may also be by injection of DNA (DNA immunization). For this purpose, the target gene is first cloned into an expression vector, so that the target sequence is under the control of a strong eukaryotic promoter (eg CMV promoter). Subsequently, DNA (eg 1-10 μg per injection) as an immunogen is transferred with a "gene gun" into strongly perfused, capillary areas of an organism (eg mouse, rabbit) The transferred DNA is taken up by cells of the animal, the target gene is expressed and the animal eventually develops an immune response against the target protein (Jung et al., Mol. Cells 12: 41-49, 2001; Kasinrerk et al., Hybrid Hybridomics 21: 287-293, 2002).

Obtaining monoclonal antibody

monoclonal antibody are traditionally made with the help of the Hybridoma technology (Technical Details: see "Monoclonal Antibodies: A Practical Approach "by Philip Shepherd, Christopher Dean ISBN 0-19-963722-9; "Antibodies: A Laboratory Manual "by Ed Harlow, David Lane ISBN: 0879693142, "Using Antibodies: A Laboratory Manual: Portable Protocol NO "by Edward Harlow, David Lane, Ed Harlow ISBN: 0879695447). As a new method is also the so-called "SLAM" technology used B cells isolated from whole blood and the cells monoclonalized. Subsequently becomes the supernatant the isolated B-cell analyzed for their antibody specificity. In contrast to hybridoma technology, the variable region of the antibody gene amplified by a single cell PCR and cloned into a suitable vector. In this way, the recovery of monoclonal antibodies is accelerated (de Wildt et al., J. Immunol., Methods 207: 61-67, 1997).

5. Validation the targets with protein-chemical methods using antibodies

With the antibodies, which can be produced as described above, can be a Make a series of important statements about the target protein. In detail are the following analyzes for the validation of the target protein sense:

Specificity of the antibody

Around to show that an antibody specifically only to the desired Target protein binds, is best suited to cell culture-based Tests with following Western Blot (various variations are described, for example, in "Current Protocols in Protein Chemistry ", John Wiley & Sons Ltd., Wiley InterScience). For detection, cells with a cDNA for transfected the target protein under the control of a strong eukaryotic promoter (e.g., cytomegalovirus promoter; CMV). The transfection of cell lines with DNA are the most diverse Methods (e.g., electroporation, liposome-based transfection, calcium phosphate precipitation) well established (e.g., Lemoine et al., Methods Mol. Biol. 75: 441-7, 1997). Alternatively you can Cell lines expressing the target gene endogenously can also be used (Proof of Target gene-specific RT-PCR). To control are in the experiment ideally homologous genes are transfected with, in the following Western Blot the specificity of the analyzed antibody to be able to prove.

in the subsequent Western Blot are cells from cell culture or tissue samples that could contain the target protein, in a 1% SDS solution lysed and thereby denaturing the proteins. The lysates will be on 8-15% denaturing polyacrylamide gels (contain 1% SDS) electrophoretically in size separated (SDS-polyacrylamide gel electrophoresis, SDS-PAGE). Then be the proteins by one of several blotting methods (e.g., semi-dry electroblotted; Biorad) to a specific membrane (e.g. Nitrocellulose, Schleicher & Schull). On This membrane can do the desired Protein be visualized. For this purpose, the membrane is first with the antibody, which recognizes the target protein (dilution about 1: 20-1: 200, depending on specificity the antibody), for 60 Incubated for a few minutes. After a washing step, the membrane is with a second, with a marker (e.g., enzymes such as peroxidase or alkaline phosphatase) coupled antibody, the first Detects antibodies. In a color or chemiluminescent reaction, the Target protein on the membrane (e.g., ECL, Amersham Bioscience). An antibody with a high specificity for the Target protein should ideally recognize only the desired protein itself.

Localization of the target protein

to confirmation the one in the "in silico "approach identified Membrane localization of the target protein will be different procedures used. An important and well established method using The antibody described above is immunofluorescence (IF). For this cells of established cell lines are used, which either synthesize the target protein (detection of the RNA in the RT-PCR or of the protein in Western blot) or transfected with plasmid DNA have been. The transfection of cell lines with DNA are the most diverse Methods (e.g., electroporation, liposome-based transfection, calcium phosphate precipitation) well established (e.g., Lemoine et al., Methods Mol. Biol. 75: 441-7, 1997). The transfected into the cells plasmid can in immunofluorescence encode the unmodified protein or even different amino acid markers couple to the target protein. The most important markers are e.g. the fluorescent "green fluorescent protein "(GFP) in its different differentially fluorescent forms, short Peptide sequences of 6-12 amino acids for which high affine and specific antibodies to disposal or the short amino acid sequence Cys-Cys-X-X-Cys-Cys, the above their cysteines can bind specific fluorescent substances (Invitrogen). Cells that synthesize the target protein become e.g. fixed with paraformaldehyde or methanol. Subsequently, the Cells on demand by incubation with detergents (e.g., 0.2% Triton X-100) be permeabilized. Subsequently, the cells with a primary antibody incubated against the target protein or against one of the coupled Marker is directed. After a washing step, the approach with a second, with a fluorescent marker (e.g., fluorescein, Texas Red, Dako) coupled antibody incubated at the first antibody binds. Subsequently the cells marked in this way are overlaid with glycerol and with the help of of a fluorescence microscope according to the manufacturer's instructions. Specific fluorescence emissions are thereby, depending on the substances used, achieved by specific excitation. The analysis usually allows the safe localization of the target protein, being for confirmation the antibody quality and the Target protein in double stains additionally the target protein also the coupled amino acid marker or other marker proteins stained whose localization has already been described in the literature is. A special case is the CFP and its derivatives, which are direct can be stimulated and even fluoresce. The membrane permeability caused by the use of detergents can be controlled, allows in immunofluorescence the demonstration, whether an immunogenic epitope is located inside or outside the cell is. The prediction the selected one Proteins can thus be underpinned experimentally. Alternatively, you can the detection of extracellular domains done by flow cytometry. These cells are under non-permeabilizing conditions (e.g., with PBS / Na azide / 2% FCS / 5 mM EDTA) and in the flow cytometer according to the manufacturer analyzed. Only extracellular Epitopes can recognized in this method by the antibody to be analyzed become. In contrast to immunofluorescence, by use from e.g. Propidium iodide or trypan blue between dead and living Cells are distinguished and thus false positive results be avoided.

One further important evidence is provided by immunohistochemistry (IHC) on specific tissue samples. The aim of this procedure is to Localization of a protein in a functionally intact tissue association to identify. The IHC serves in detail to (1) the amount to be able to estimate target protein in tumor and normal tissues, (2) to analyze how many cells in tumor and healthy tissue synthesize the target gene, and (3) to define the cell type in a tissue (tumor, healthy cells), in which the target protein is detectable. Alternatively, the Protein amounts of a target gene by tissue immunofluorescence using Digital camera and appropriate software (e.g., Tillvision, Till-photonics, Germany). The technology is widely published been, details for coloring and Microscopy is therefore e.g. "Diagnostic Immunohistochemistry" by David J., MD Dabbs ISBN: 0443065667 or in "Microscopy, Immunohistochemistry, and Antigen Retrieval Methods: For Light and Electron Microscopy "ISBN: 0306467704 refer to. It should be noted that due to the properties of antibodies different protocols must be used (below is an example described) in order to come to a meaningful result.

In usually histologically defined tumor tissue and as a reference comparable healthy tissue used in the IHC. As a positive and negative controls can It also serve cell lines in which the presence of the target gene by RT-PCR Analyzes is known. A background check is always to be carried.

Formalin-fixed (another fixation method with eg methanol is also possible) and paraffin-embedded pieces of tissue with a thickness of 4 .mu.m are applied to a glass substrate and, for example, deparaffinized with xylene. The samples are washed with TBS-T and blocked in serum. This is followed by incubation with the first antibody (dilution: 1: 2 to 1: 2000) for 1-18 hours, affinity-purified antibodies being generally used. After a washing step, a 30-60 minute incubation with a second antibody, which is carried out with an alkaline phosphatase (Alternatively: eg peroxidase) is coupled and directed against the first antibody. Subsequently, a color reaction is carried out using alkaline phosphatase (see, for example, Shi et al., J. Histochem Cytochem., 39: 741-748, 1991, Shin et al., Lab. Invest., 64: 693-702, 1991). For detection of antibody specificity, the reaction can be competed by prior addition of the immunogen.

Analysis of protein modifications

Secondary protein modifications such as N- and O-glycosylations or myristilations can the accessibility inhibit or even completely prevent immunogenic epitopes and thus the efficacy of antibody therapies question. In addition, it has been proven many times that Type and amount of secondary modifications in normal and tumor tissues (e.g., Durand & Seta, 2000; Clin. Chem. 46: 795-805; Hakomori, 1996; Cancer Res. 56: 5309-18). The Analysis of these modifications is therefore essential for therapeutic success an antibody. Potential binding sites can be determined by specific algorithms predict.

The Analysis of protein modifications usually occurs in Western Blot (see above). Especially glycosylations, which in the Usually a size of several kDa have lead to a larger total mass of the target protein, which can be separated in SDS-PAGE. To the Detection of specific O- and N-glycosidic bonds Protein lysates prior to denaturation by SDS with O- or N-glycosylases incubated (according to the manufacturer's instructions, for example PNgase, Endoglycosidase F, endoglycosidase H, Roche Diagnostics). Then done a Western blot as described above. In reducing the Size of one Target protein can be so after incubation with a glycosidase detected specific glycosylation and in this way also the tumor specificity a modification are analyzed.

Functional analysis of the target gene

The Function of the target molecule can be crucial for its therapeutic benefit, allowing functional analysis an important building block in the characterization of therapeutic usable molecules are. The functional analysis can be done either in cells in cell culture experiments or in vivo using animal models. This is the Gene of the target molecule either knocked out by mutation or the target sequence into the cell or the organism inserted ("knockin") Changes in the cellular Context on the one hand by the loss of function of the analyzed Genes ("loss of function "). In the second case, changes can be made analyze that by the supplement caused by the analyzed gene ("gain of function").

a. Functional analysis in cell

Transfection. To analyze the "gain of function "must the gene of the target molecule be transferred to the cell. These are cells with a DNA tansfiziert, which allow the synthesis of the target molecule. Usually stands the gene of the target molecule thereby under the control of a strong eukaryotic promoter (e.g. Cytomegalovirus promoter; CMV). For transfection of cell lines with DNA are a variety of methods (e.g., electroporation, on Liposome-based transfection, calcium phosphate precipitation) well established (e.g., Lemoine et al., Methods Mol. Biol. 75: 441-7, 1997). The gene can either transient without genomic integration or with genomic integration after selection with e.g. neomycin be stably synthesized.

RNA interference (siRNA). An expression inhibition of the target gene, the in certain circumstances a complete one Functional loss of the target molecule in Cells induced by the "RNA interference" (siRNA) technology in cells (Hannon, GJ 2002. RNA interference 418: 244-51; Czauderna et al. 2003. Nucl. Acid Res. 31: 670-82). These are cells with short, about 20-25 nucleotides long, double-stranded RNA Molecules transfected, the for the target molecule are specific. An enzymatic process subsequently leads to degradation the specific RNA of the target gene, resulting in decreased expression of the target protein, thus enabling the functional Analysis of the target gene.

Cell lines which were modified by means of transfection or siRNA, can subsequently different way to be analyzed. Below are the most common Examples listed.

1. Proliferation and cell cycle behavior

A large number of methods are established for the analysis of cell proliferation and are commercially available from various companies (eg Roche Diagnostics, Invitrogen, details of the test methods are described in the numerous application protocols). The cell count in cell culture experiments can be determined by simple counting or by colometric tests that measure the metabolic activity of the cells (eg, wst-1, Roche Diagnostics). Metabolic test methods indirectly measure the number of cells in an experiment via enzymatic markers. Direct cell proliferation by analysis of the DNA synthesis rate eg can be measured by addition of bromodeoxyuridine (BrdU), the detection of the integrated BrdU is carried out by specific antibodies using a colometric method.

2. Apoptosis and cytotoxicity

A size Number of assay systems for detecting cellular apoptosis and cytotoxicity are available. One the crucial characteristic is the specific, enzyme-dependent fragmentation the genomic DNA that is irreversible and sure to kill the Cell leads. Method for detecting these specific DNA fragments are commercially available. As an additional Procedure is the "TUNEL assay ", the DNA single-strand breaks can also detect in tissue sections. Cytotoxicity is present everything over a changed one cell permeability detected as markers for the vitality state of cells. These are either in cell culture supernatant Analyzes markers that are normally found intracellularly. alternative can also absorb capacity be analyzed by color markers that are not recorded by intact cells become. The best known examples of color markers are trypan blue and propidium iodide, a common one intracellular Marker is the lactate dehydrogenase, which is detected enzymatically in the supernatant can be. Different test systems are different commercial suppliers (e.g., Roche Diagnostics, Invitrogene) Available.

3. Migration assay

The ability cells to migrate is in a specific migration test preferably analyzed using a Boyden chamber (Corning Costar) (Cinamon G., Alon R.J. Immunol., Methods, 2003 Feb; 273 (1-2): 53-62; Stockton et al. 2001. Mol. Biol. Cell. 12: 1937-56). To do this Cells on a filter with specific pore size cultivated. Cells that can migrate are able to pass through this filter into another underneath Hiking culture vessel. A subsequent one Microscopic analysis then allows the determination of one possibly changed Migration behavior that is due to the "gain of function" or "loss of Function "of the target molecule induced has been.

b. Functional analysis in animal models

alternative Cell culture experiments offer an analysis of the target gene function elaborate in vivo experiments in animal models. These models have Compared to the cell-based method the advantage that they can prove aberrations or diseases that only in the context of the whole organism are detectable. A variety of models for Human diseases are now available (Abate-Shen & Shen. 2002. Trends in Genetics S1-5; Matsusue et. al. 2003. J. Clin. Invest. 111: 737-47). Various animal models such as yeast, nematodes or zebrafish are been intensively characterized. Preferred models are but compared to other species mammal animal models such as Example, the mouse (Mus musculus) because they are the biological processes in the humane context. For mice are in recent years Both transgenic methods have been established, the new genes in the Integrate mouse genome ("gain of function "; Jegstrup I. et al. 2003. Lab Anim. 2003 Jan.; 37 (1): 1-9). Alternatively through other methodological approaches Genes in the mouse genome turned off and so a loss of function of a desired Gene induces (knockout models, "loss of function"; Zambrowicz BP & Sands AT. Nat. Rev. Drug Discov. 2003 Jan; 2 (1): 38-51; Niwa H. 2001. Cell Struct. Funct. 2001 Jun; 26 (3): 137-48.); technical details are widely published.

To Generation of mouse models can change, by the transgene or by the loss of function of a gene be analyzed in the context of the whole organism (Balling R, 2001 Ann. Rev. Genomics Hum Genet 2: 463-92). So are for example behavioral tests as well as biochemical investigations established blood parameters possible. Histological analysis, immunohistochemistry or electron microscopy enable the characterization of changes cellular Level. The specific expression pattern of a gene can by In situ hybridization can be detected (Peters T. et al. 2003. Hum. Mol. Genet 12: 2109-20).

Example 1: Identification SEQ ID NO: 1/2 as a diagnostic and therapeutic cancer target

The SEQ ID NO: 1 (nucleic acid sequence) is encoded and represented by a new gene on chromosome 6 (6q26-27) the deduced protein sequence (SEQ ID NO: 2). An alternative open reading frame of this gene locus is SEQ ID NO: 267, which is derived for the Protein sequence SEQ ID NO: 268 encoded. Both protein sequences show no homologies to already known proteins.

According to the invention, after the establishment of a specific quantitative RT-PCR (primer pair SEQ ID NO: 3 and 4), the amount of gene-specific transcripts in healthy tissue and in carcinoma samples was investigated. The transcript could not be detected in any of the normal tissues analyzed. Surprisingly, we specifically detected substantial amounts of this transcript in almost all melanoma samples studied, although the gene is not expressed in normal skin as the source tissue ( 1 ). By a conventional RT-PCR the selectivity of this marker for melanomas was confirmed. ( 2 ). Surprisingly, we amplified two fragments that reflect gene-specific variants (probably SEQ ID NO: 1 and SEQ ID NO: 267).

We show that this gene is an absolutely specific marker for melanoma cells is and by its absence in each examined normal tissue for purposeful Therapy and diagnostics approaches is suitable as a biomarker.

Especially extracellular According to the invention, proportions of SEQ ID NO: 2 or 268 can serve as the target structure of monoclonal antibodies be used. This affects u.a. following epitopes: amino acids 1-50, based on SEQ ID NO: 2; Amino acids 1-12, based on the SEQ ID NO: 268, amino acids 70-88 with reference to SEQ ID NO: 2, amino acids 33-129 based on SEQ ID NO: 268, and SEQ ID NO: 281.

According to the invention are therapeutic also other goal-oriented approaches like vaccines and therapies with "small compounds "conceivable, which only have this gene as the target structure and therefore no healthy ones Affect cells. Also diagnostically, this gene may be due to its selectivity used for tumor cells become.

Example 2: Identification SEQ ID NO: 5/6 as a diagnostic and therapeutic cancer target

The SEQ ID NO: 5 (nucleic acid sequence) is encoded and represented by a new gene on chromosome 11 (11 q 12.1) the deduced protein sequence (SEQ ID NO 6). An alternative open reading frame this gene locus is SEQ ID NO: 269, which is for the deduced protein sequence SEQ ID NO: 270 encoded. Both protein sequences show no homologies to already known proteins.

According to the invention, after the establishment of a gene-specific quantitative RT-PCR (primer pair SEQ ID NO: 7 and 8), the amount of gene-specific transcripts in healthy tissue and in carcinoma samples (each pool of samples) was investigated. With the exception of testis, we did not detect any specific RNA or only in small amounts in the healthy tissues we examined ( 3 ; A: quantitative RT-PCR; B: gel image). The locus thus expresses a germ cell specific gene product with stringent suppression of expression in normal somatic tissues. However, the gene is activated in many tumor samples, specific RNA was detectable in substantial amounts ( 3 and 4 ). The highest prevalence and expression levels were found in renal cell tumors. However, specific transcripts were also detectable in gastric, pancreatic, ENT and lung tumors. To additionally demonstrate the expression of this gene locus, a Northern blot was additionally performed. For this purpose, a probe was prepared in a specific PCR of primers SEQ ID NO: 7 and 8 incorporating digoxygenin-dUTP (Roche Diagnostics) according to the manufacturer's instructions. The probe was then incubated with 2 μg ( 5a , Lane 1) or 1 μg ( 5a , Lane 2) total RNA from testis tissue hybridized and the digoxygenin the probe subsequently detected in a specific color reaction. An approximately 3.1 kb gene-specific fragment could be detected in the experiment ( 5a ) and thus additionally confirmed the expression of this locus.

Of the Genlokus is thus a typical representative of the class of so-called. Cancer / testis antigens, in normal tissues almost exclusively in the germ cells of the Testis are expressed. In tumors, however, become cancer / testis antigens often even though they are in the underlying somatic normal tissue cells not expressed. Several members of this functional and structurally heterogeneous class are due to their attractive selective tissue distribution already for specific immunotherapeutic approaches in cancers in Phase I / II studies (e.g., Scanlan MJ, Gure AO, Jungbluth AA, Old LJ, Chen YT. 2002. Immunol Rev. 2002 Oct; 188: 22-32).

For the production of antibodies, among others, the peptides SEQ ID NO: 282 and 283 can be used. In particular, the extracellular domains of SEQ ID NO: 6 or SEQ ID NO: 270 can be used according to the invention as the target structure of monoclonal antibodies. The immunization of animals with these peptides resulted in the availability of antibodies with which we were able to confirm the data of the PCR examination also at the protein level by immunohistochemical investigations. Immunohistochemical staining of testis as a positive control but also of kidney tumors ( 5b ) showed that this newly discovered gene product is indeed translated into protein, that it is not detectable in normal tissues other than testis, but in tumors, and that tumor cells of a patient are actually stained on the surface. Furthermore, these data support that this gene product with its extraordinary tumor cell selectivity can serve as the target structure for a therapeutically and diagnostically usable antibody, as well as that in principle such antibodies can be generated against this gene product.

Example 3: Identification of LOC203413 as a diagnostic and therapeutic cancer target

The gene or protein of the gene locus LOC203413 (nucleic acid sequence: SEQ ID NO: 9; Amino acid sequence: SEQ ID NO 10) is a hitherto uncharacterized gene on the X chromosome (Xq24). Apart from a transmembrane domain, it has no other functional motifs and no homologies to previously known proteins.

According to the invention, after the establishment of a LOC203413-specific quantitative RT-PCR (primer pair SEQ ID NO: 11 and 12), the amount of transcript in healthy tissue and in carcinoma samples ( 6a , b, c). LOC203413-specific RNA, with the exception of testis, can not be detected in any of the healthy tissues we studied. LOC203413 is therefore a germ cell-specific gene product whose transcription is stringently suppressed in normal somatic tissues. As 6a , b, c, LOC203413-specific transcripts were detectable in tumors of certain organs, particularly in the stomach, pancreatic, esophageal, mammary, ovarian and prostate cancers, although they are undetectable in corresponding normal tissues. The incidence of patients carrying this marker is between 40-70%, depending on the tumor type.

LOC203413 is thus a typical representative of the class of so-called. Cancer / testis antigens, those in normal tissues exclusively are expressed in the germ cells of the testis. In tumors, however Cancer / Testis antigens are common even though they are in the underlying somatic Normal tissue cells are not expressed. Several members of this Functionally and structurally heterogeneous class are due to their attractive selective tissue distribution already for specific immunotherapeutic approaches in cancers in Phase I / II studies (e.g., Scanlan MJ, Gure AO, Jungbluth AA, Old LJ, Chen YT. 2002. Immunol Rev. 2002 Oct; 188: 22-32).

Especially the extracellular domain of LOC203413 can be used according to the invention as a target structure of monoclonal antibodies be used. Thus, amino acids 22-113 (SEQ ID NO: 284) interesting as epitopes. In the sequence, with reference to SEQ ID NO: 10 at amino acid position 34 and localized 83 conserved N-glycosylation motifs that are in certain circumstances especially for the production of tumor-specific antibodies are suitable. For the production of LOC203413-specific antibodies the peptides listed under SEQ ID NO: 285 and 286 were used.

We have been able to demonstrate that such antibodies can specifically stain cells which are transfected and express the said gene product ( 7 ). This supports that this gene product can be detected in tumor cells and is suitable as a target structure for a therapeutically and diagnostically usable antibody, as well as that in principle such antibodies can be generated against this gene product. According to the invention, other target-oriented approaches such as vaccines and therapies with "small compounds" are conceivable therapeutically, which have only this gene as the target structure and thus do not affect healthy cells. "Also diagnostically, this gene can be used for tumor cells due to its selectivity.

Example 4: Identification of LOC90625 as a diagnostic and therapeutic cancer target

The Gen LOC90625 (nucleic acid sequence: SEQ ID NO: 13) is a hitherto uncharacterized gene on chromosome 21 (21q22.3). It codes for a protein (amino acid sequence: SEQ ID NO: 14) with a transmembrane domain but otherwise no homologies to already known proteins.

According to the invention, after the establishment of a LOC90625-specific quantitative RT-PCR (primer pair SEQ ID NO: 15 and 16), the amount of gene-specific transcripts in healthy tissue and in carcinoma samples was investigated ( 8a , b). LOC90625 is very selectively expressed in healthy tissue, specific transcripts are detectable especially in the testis. Little or no detectable LOC90625-specific expression in all other healthy tissues analyzed ( 8a , b).

Surprisingly, we detected LOC90625-specific overexpression in various tumor types. In particular in prostate, esophageal and pancreatic carcinomas, LOC90625 was highly overexpressed compared to the respective healthy tissue samples ( 8a , b)

LOC90625 is a selectively expressed antigen that is evident in proliferating Reinforced tissues is expressed. This selective overexpression in tumors is can be used diagnostically and therapeutically.

Especially the extracellular domain of LOC90625 can be used according to the invention as a target structure of monoclonal antibodies be used. This can e.g. 1-19 (SEQ ID NO: 287) or amino acids 40-160 (SEQ ID NO: 288) be. For the preparation of LOC203413-specific antibodies were the peptides according to SEQ ID NO: 289 and 290 used.

We have been able to demonstrate that such an antibody can specifically stain cells which are transfected and express the said gene product ( 9a ). Also, this antibody clearly has a protein of the correct size in the Western blot ( 9b ). Immunohistochemical staining of testis as a positive control but also of Prostate tumors ( 9c ) showed that this newly discovered gene product is indeed translated into protein, that it is not detectable in normal tissues other than testis, but in tumors, and that tumor cells of a patient are actually stained on the surface. Furthermore, these data support that this gene product with its extraordinary tumor cell selectivity can serve as the target structure for a therapeutically and diagnostically usable antibody, as well as that in principle such antibodies can be generated against this gene product.

According to the invention are therapeutic also other goal-oriented approaches like vaccines and therapies with "small compounds "conceivable, which only have this gene as the target structure and therefore no healthy ones Affect cells. Also diagnostically, this gene may be due to its selectivity used for tumor cells become.

Example 5: Identification of the protein FAM26A as a diagnostic and therapeutic cancer target

The Gene FAM26A (SEQ ID NO: 17; NM_182494) located on chromosome 10 (10q24) is located, encodes the gene product of SEQ ID NO: 18 (NP_872300). FAM26A has multiple transmembrane domains. At amino acid position 142 is a motive for an N-glycosylation localized.

According to the invention, after the establishment of a FAM26A-specific quantitative RT-PCR (primer pair SEQ ID NO: 19 and 20), the amount of gene-specific transcripts in healthy tissue and in tumor samples was investigated ( 10 ). Surprisingly, we were able to demonstrate the overexpression of FAM26A in various tumors. Especially in ovarian, gastric, esophageal, pancreatic and hepatic tumors, FAM26A was significantly more expressed compared to the corresponding healthy tissue. The selectively high expression of FAM26A in various tumor tissues can be used according to the invention for molecular diagnostic methods such as RT-PCR for the detection of tumor cells in tissue biopsies.

FAM26A-specific antibodies were prepared by immunization of animals using, inter alia, the peptides listed under SEQ ID Nos. 291 and 292. The specificity of the antibodies was demonstrated by Western blot analysis in COS cells which had been transfected with a plasmid construct coding for a FAM26 fragment ( 11 ). Western blot analysis also revealed clear overexpression in various cervical, ovarian and pancreatic orbital biopsies of various patients ( 12 ), as well as in the RT-PCR-positive cell lines SW480, EFO 27 and SNU 16 ( 13 ) detectable. In addition to an expected 40 kDa protein band, we also found an approximately 50 kDa specific band, which represents a posttranslationally modified protein.

The endogenous FAM26A protein could be detected in SW480 cells by immunofluorescence using a SEQ ID NO: 291 specific antibody. The analysis shows a localization in the plasma membrane ( 14 ). By immunohistochemical staining with the antibody, we also detected the gene product in patient material ( 15 : A pancreatic tumor, B testis). Immunohistochemical staining of testis as a positive control but also of pancreatic tumors has revealed that this newly discovered gene product is indeed translated into protein, that it is not detectable in normal tissues other than testis, but in tumors, and that tumor cells of a patient are actually stained on the surface. Furthermore, these data support that this gene product with its extraordinary tumor cell selectivity can serve as the target structure for a therapeutically and diagnostically usable antibody, as well as that in principle such antibodies can be generated against this gene product. In particular, the extracellular domains of FAM26A can be used according to the invention as target structures of monoclonal antibodies. With reference to SEQ ID NO: 17, these are amino acids 38-48 (SEQ ID NO: 293) and amino acids 129-181 (SEQ ID NO: 294). Alternatively, C-terminal amino acids 199-334 (SEQ ID NO: 295) could also be preferred epitopes for the production of antibodies for diagnostic or therapeutic purposes. In addition, the N-glycosylation motif at position 142 may be an interesting target for therapeutic antibodies.

Also according to the invention therapeutically, other goal-oriented approaches such as vaccines and therapies with "small compounds "conceivable, which only have this gene as the target structure and therefore no healthy ones Affect cells. Also diagnostically, this gene may be due to its selectivity for tumor cells be used.

Example 6: Identification of SEMA5B as a diagnostic and therapeutic cancer target

The Gene Semaphorin 5B (SEMA5B; SEQ ID NO: 21) that the protein of the SEQ ID NO: 22 is located on chromosome 3 (3q21.1). SEMA5B is a type I transmembrane protein and heard to the family of semaphorins.

According to the invention, after the establishment of a SEMA5B-specific quantitative RT-PCR (primer pair SEQ ID NO: 23 and 24), the amount of gene-specific transcripts in healthy tissues and carcinoma samples ( 16 ). We found SEMA5B in healthy tissue very selectively restricted to testis and skin. Little or no detectable SEMA5B-specific expression in all other healthy tissues analyzed ( 16 ). In contrast, we found a SEMA5B-specific overexpression in some types of tumors, especially in renal and breast tumors, compared to their healthy tissues. ( 17A , B).

The selective overexpression in tumors is therapeutically usable.

Especially the extracellular domain of SEMA5B (amino acids 20-1035; SEQ ID NO: 296) can be used according to the invention as a target structure of antibodies become. SEMA5B is a type I transmembrane domain protein (TM amino acids 1035-1057), whose C-terminus is located inside the cell (amino acids: 1058-1151). For making SEMA5B-specific antibody were the peptides according to SEQ ID NO: 297 and 298 used.

Example 7: Identification of GJB5 as a diagnostic and therapeutic cancer target

The Protein GBJ5 (nucleic acid sequence: SEQ ID NO: 25; Amino acid sequence: SEQ ID NO: 26) is a member of the connexin family. The gene exists from two exons and is located on chromosome 1 (1p35.1). The derived amino acid sequence encodes for a protein with 273 amino acids. Connexins have an important function in cell-cell contacts via so-called "gap junctions" that exchange of small cytoplasmic molecules, ions and secondary transmitters serve and thus the communication between individual cells enable. Gap junctions consist of several connexin subunits, the to form a membrane channel. So far, 11 different members of the Connexins are described, all in a genome on chromosome 1 (Richard, G., Nature Genet 20: 366-369, 1998). GBJ5 has four transmembrane domains, the N- and C-terminus of the protein are located inside the cell.

According to the invention, after establishing a GBJ5-specific quantitative RT-PCR (primer pair SEQ ID NO: 27, 28), the amount of gene-specific transcripts in healthy tissue and in carcinoma samples was investigated (pool of samples, the number is shown in the figure). Our investigations show a differential distribution of expression in normal tissues. GBJ5 transcripts were found to be expressed almost exclusively in the esophagus and in the skin, in all other tissues analyzed, transcription is very weak or undetectable ( 18a ). Surprisingly, we found that various tumor tissues (including esophageal, colon, gastric, lung, cervical, head / neck and pancreatic carcinomas) show a strong overexpression of this gene product compared to the respective normal tissues ( 18a , b, c, d).

In particular, the extracellular domains of GBJ5 can be used according to the invention as the target structure of therapeutic antibodies. Referring to SEQ ID NO: 26, amino acids 41-75 (SEQ ID NO: 299) and the region between amino acids 150 and 187 (SEQ ID NO: 300) are located extracellularly. For the production of GJB5-specific antibodies, the peptides were used with the SEQ ID NO: 301 and 302 and led to the successful production of specific antibodies that recognize only the corresponding molecule in the Western blot ( 19 ).

Example 8: Identification of KLK5 as a diagnostic and therapeutic cancer target

The Gene KLK5 (SEQ ID NO: 29) and its translation product (SEQ ID NO: 30) is a member of the kallikrein family, a group of serine proteases with different physiological functions. The gene is on chromosome 19 (19q13.3-13.4) and encodes a serine protease. KLK5 is synthesized as a proform and in the stratum corneum by proteolysis activated. (Brattsand, M et al; J. Biol. Chem. 274: 1999). The active one Protease (amino acids 67-293) is secreted and is in the process of skin assessment involved. The propeptide (amino acids 30-67) remains over the transmembrane domain (Amino acids 1-29) bound to the cell surface (Ekholm, E et al; Jour Investigative Dermatol, 114, 2000).

According to the invention, after establishing a KLK5-specific quantitative RT-PCR (primer pair SEQ ID NO: 31, 32), the selective distribution of KLK5-specific transcripts in carcinoma samples was investigated in comparison to healthy tissue ( 20 ). In most normal tissues, KLK5 is very low to none expressed, whereas moderate expression of KLK5 was found only in testis, esophagus, skin and prostate. Significant overexpression of KLK5 to the corresponding normal tissues of origin has been detected in tumors, namely esophageal carcinoma, cervix, and in ENT tumors ( 20 ). A markedly weaker, but detectable KLK5-specific expression could also be detected in some tumors of other tissues (eg in gastric and pancreatic carcinomas).

In particular, the extracellular domain of KLK5 can be used according to the invention as the target structure of therapeutic antibodies (SEQ ID NO: 303). Part of this domain is cleaved off after secretion of the protein to the cell surface and secreted. This portion of the protein is less suitable as an epitope for therapeutic antibodies than preferably the region of the propeptide (amino acids 30-67) which remains anchored to the cell membrane after processing of the portion to be secreted. For the production of KLK5-specific antibodies, the peptide according to SEQ ID NO: 304 were used. Western blot shows the specificity of this peptide for the sequence of the gene product KLK5 ( 21a ). This antibody also stains tumors in immunohistochemistry. This is exemplified for breast tumors in which this protein is highly enriched while in normal mammary tissue no protein is detectable ( 21b ).

These Support data, that this gene product actually translates to protein that it is not in normal tissues other than testis, but is detectable in tumors, and that tumor cells of a patient indeed on the surface to be stained. Further support this data that this gene product with its extraordinary Tumor cell selectivity as target for a therapeutic and diagnostically useful antibodies, as well as that in principle such antibodies against this gene product can be generated.

Example 9: Identification of LOC352765 as a diagnostic and therapeutic cancer target

Of the Genlokus LOC352765 is located on chromosome 9 (9q34.12) Gene (SEQ ID NO: 33) encodes the gene product of SEQ ID NO: 34 LOC352765 protein has a transmembrane domain at the N-terminus. The hypothetical Protein shows no homologies to already known proteins.

According to the invention, after the establishment of a LOC352765-specific quantitative RT-PCR (primer pair SEQ ID NO: 35 and 36), the amount of gene-specific transcripts in healthy tissue and in carcinoma samples (pool of samples) was investigated ( 22 ). LOC352765 is very selectively expressed in healthy tissue and specific transcripts were found only in the testis, skin and bladder. In contrast, LOC352765-specific overexpression could be detected in some tumor types. Especially in breast tumors the expression was higher than that of the most expressive normal tissue ( 22 . 23 ). Also in colon and ovarian carcinomas and in ENT tumors we found LOC352765 clearly overexpressed.

LOC352765 is due to its selective overexpression therapeutically usable in tumors. In particular, the extracellular domain of LOC352765 (Amino acids 44-211, SEQ ID NO: 34) can be used according to the invention as a target structure of antibodies and other targeted therapies are used. For the production of specific antibodies was the peptides according to SEQ ID NO: 305 and 306 used.

Example 10: Identification of SVCT1 as a diagnostic and therapeutic cancer target

The Gene SVCT1 (SEQ ID NO: 37) is located on chromosome 7 (7q33) and encodes for the gene product of SEQ ID NO 38. The SVCT1 protein has four transmembrane domains and shows no homologies to already known proteins.

According to the invention, after the establishment of an SVCT1-specific quantitative RT-PCR (primer pair SEQ ID NO: 39 and 40), the amount of gene-specific transcripts in healthy tissue and in carcinoma samples (pool of samples) was investigated ( 24 ). SVCT1 is selectively restricted to kidney, testis, thymus and mammary gland in healthy tissues. Surprisingly, SVCT1-specific overexpression could be detected in some tumor types. Particularly in kidney, cervical, esophageal and pancreatic carcinomas and in ENT tumors SVCT1 is highly overexpressed ( 24 . 25A , B) not only compared to the corresponding healthy tissue of origin, but also in relation to the most expressive normal tissue ever.

SVCT1 is due to its selective overexpression therapeutically usable in tumors. In particular, the extracellular domains of SVCT1 can according to the invention as a target structure of antibodies or other targeted therapies. to Production of specific antibodies For example, the peptides of SEQ ID NOs: 307 and 308 were used.

Example 11: Identification of LOC199953 as a diagnostic and therapeutic cancer target

The Gene or protein of gene locus LOC199953 (nucleic acid sequence: SEQ ID NO: 41; Amino acid sequence: SEQ ID NO: 42) is located on chromosome 1 (1q36.22). The protein has several transmembrane domains. Alternative open reading frames of this gene locus represent the SEQ ID NO: 271 with its gene product SEQ ID NO: 272 and SEQ ID NO: 273 with the associated Gene product SEQ ID NO: 274. In addition, the hypothetical Protein no further homologies to already known protein domains.

According to the invention was after Etablie a LOC199953-specific quantitative RT-PCR (primer pair SEQ ID NO: 43 and 44) the amount of gene-specific transcripts in healthy tissue and in carcinoma samples examined. LOC 199953 is selectively expressed in healthy tissues and overexpressed in some tumors. In particular, in ENT and renal carcinomas ( 26 ) in about 50% of the tumor samples overexpression compared to normal tissues are identified.

The extracellular domains from LOC199953 according to the invention as a target structure of antibodies be used.

Example 12: Identification of TMEM31 as a diagnostic and therapeutic cancer target

The Gene TMEM31 (SEQ ID NO: 45) of the gene locus LOC203562 is on chromosome X (Xq22.2) localized. The gene encodes the protein of SEQ ID NO: 46. The protein has two transmembrane domains and shows no homologies otherwise to already known proteins.

According to the invention, after the establishment of a TMEM31-specific quantitative RT-PCR (primer pair SEQ ID NO: 47 and 48), the amount of gene-specific transcripts in healthy tissue and in carcinoma samples was investigated. TMEM31 is very selectively restricted to testis in healthy tissues ( 27 ). Surprisingly, we also find expression in some tumor types (these are in particular kidney, colon, stomach, breast, liver, lung and ENT carcinomas), while in the corresponding normal tissues no expression was detectable. ( 27 . 28A , B).

TMEM31 is thus a typical representative of the class of so-called. Cancer / testis antigens, those in normal tissues exclusively are expressed in the germ cells of the testis. In tumors, however Cancer / Testis antigens are common even though they are in the underlying somatic Normal tissue cells are not expressed. Several members of this Functionally and structurally heterogeneous class are due to their attractive selective tissue distribution already for specific immunotherapeutic approaches in cancers in Phase I / II studies (e.g., Scanlan MJ, Gure AO, Jungbluth AA, Old LJ, Chen YT. 2002. Immunol Rev. 2002 Oct; 188: 22-32).

The extracellular domains from TMEM31 according to the invention as a target structure of antibodies be used.

Example 13: Identification of FLJ25132 as a diagnostic and therapeutic cancer target

The Gene / protein FLJ25132 (nucleic acid sequence: SEQ ID NO: 49; Amino acid sequence: SEQ ID NO: 50) is located on chromosome 17 (17q25.3). FLJ25132 has a transmembrane domain, otherwise show no homologies to already known proteins.

According to the invention, after the establishment of a FLJ25132-specific quantitative RT-PCR (primer pair SEQ ID NO: 51 and 52), the amount of gene-specific transcripts in healthy tissue and in carcinoma samples was investigated. FLJ25132 is partially overexpressed in the carcinoma samples we examined compared to healthy tissue ( 29 ). Particularly in ovarian and prostate carcinomas, a clear overexpression of FLJ25132 was detected.

The extracellular domains from FLJ25132 according to the invention as a target structure of antibodies be used.

Example 14: Identification from LOC143724, LOC284263, LOC283435 and LOC349260 as diagnostic and therapeutic cancer targets

The Gene loci (with the appropriately encoded genes and gene products) LOC143724, LOC284263, LOC283435 and LOC349260 are due to their similarity Profiles summarized.

The in Genlokus LOC143724 contained gene with the SEQ ID NO: 53 on chromosome 11 (11q13.1) encodes SEQ ID NO: 54 as a gene product. An alternative open reading frame of this gene locus is represented by SEQ ID NO: 275 with its gene product SEQ ID NO: 276 and represents either an independent one Transcript or a splice variant of SEQ ID NO: 53. For the gene-specific amplification of the gene were the primers according to SEQ ID NO: 55 and 56 used.

The in the gene locus LOC284263 gene containing SEQ ID NO: 89 on chromosome 18 (18q21.1) encodes the gene product with SEQ ID NO: 90. For gene-specific For amplification of the gene, the primers according to SEQ ID NO: 91 and 92 were used.

The gene contained in the gene locus LOC283435 with SEQ ID NO: 117 on chromosome 12 (12q24.32) encodes the gene product having SEQ ID NO: 118. For the gene-specific amplification of the gene, the primers according to SEQ ID NO: 119 and 120 were used , The gene contained in the gene locus LOC349260 with the SEQ ID NO: 121 on chromosome 9 (9q11.2) encodes the gene product having SEQ ID NO: 122. For the gene-specific amplification of the gene, the primers according to SEQ ID NO: 123 and 124 were used. All proteins have transmembrane domains and in addition show no homologies to already known proteins.

According to the invention was after the establishment of specific quantitative RT-PCR analyzes the amount of gene-specific transcripts in healthy tissue and examined in carcinoma samples. All four genes can be except of Testis in any of the healthy tissues we examined. The genes are therefore germ cell-specific with high probability. Surprisingly but one finds significant expression in different tumor samples.

The four genes are thus typical representatives of the class of the so-called cancer / testis antigens, those in normal tissues exclusively are expressed in the germ cells of the testis. In tumors, however Cancer / Testis antigens are common even though they are in the underlying somatic Normal tissue cells are not expressed. Several members of this Functionally and structurally heterogeneous class are due to their attractive selective tissue distribution already for specific immunotherapeutic approaches in cancers in Phase I / II studies (e.g., Scanlan MJ, Gure AO, Jungbluth AA, Old LJ, Chen YT. 2002. Immunol Rev. 2002 Oct; 188: 22-32).

The extracellular domains of the four genes can according to the invention as a target structure of antibodies be used.

Example 15: Identification SEQ ID NO: 57 as a diagnostic and therapeutic cancer target

The Sequence according to SEQ ID NO: 57 is derived from a gene on chromosome 1 (1p21.3) and encoded the protein sequence according to SEQ ID NO: 58. An alternative transcript of the gene locus is through the sequence according to SEQ ID NO: 277 with its gene product SEQ ID NO: 278. The transmembrane protein shows no homologies to already known Proteins.

According to the invention, after the establishment of a specific quantitative RT-PCR (primer pair SEQ ID NO: 59 and 60), the amount of gene-specific transcripts in healthy tissue and in carcinoma samples was investigated. SEQ ID NO: 57 is selectively expressed in the healthy tissues we studied ( 30 ). Specific transcripts were detectable in almost all tumor types analyzed and overexpressed especially in liver, ENT and kidney tumors. This was confirmed in the analysis of individual tumor samples compared to healthy tissue samples ( 31A -D).

The extracellular domains of SEQ ID NO: 58 according to the invention as a target structure of antibodies in particular, are amino acids 20-38 and 90-133 extracellular localized.

Example 16: Identification of LOC119395 as a diagnostic and therapeutic cancer target

The gene contained in gene locus LOC119395 on chromosome 17 (17q25.3) SEQ ID NO: 61 encodes a gene product having SEQ ID NO: 62. The transmembrane protein shows no homologies to already known Proteins.

According to the invention, after the establishment of a LOC119395-specific quantitative RT-PCR (primer pair SEQ ID NO: 63 and 64), the amount of gene-specific transcripts in healthy tissue and in carcinoma samples was investigated ( 32 ). LOC119395 is very selectively expressed in the healthy tissues we studied and can only be detected in a few tissues ( 32 ). In contrast, LOC119395 specific transcripts were detectable in almost all tumor types analyzed. In particular, in gastric, ovarian and prostate carcinomas, a partly clear tumor-selective overexpression of LOC119395 was observed. This was confirmed in the analysis of individual tumor samples compared to healthy tissue samples ( 33A -C). Compared to the respective healthy tissue, overexpression of LOC119395 was detected in breast carcinomas and in esophageal tumors. Tumor-selective expression could be identified in colon and gastric carcinomas ( 33 C).

The extracellular domain of LOC119395 (amino acids 44-129) can according to the invention as a target structure of antibodies be used.

Example 17: Identification of LOC121838 as a diagnostic and therapeutic cancer target

The located in gene locus LOC121838 on chromosome 13 (13q14.11) gene with the transcript of SEQ ID NO: 65, the protein encodes SEQ ID NO: 66. The transmembrane protein shows no homologies to already known proteins.

According to the invention, after the establishment of a LOC121838-specific quantitative RT-PCR (primer pair SEQ ID NO: 67 and 68), the amount of gene-specific transcripts in healthy tissue and in carcinoma samples was investigated ( 34A ). LOC121838 is very selectively expressed in the healthy tissues we studied and can only be detected in a few tissues ( 34A and B). In contrast, LOC121838 specific transcripts were detectable in many tumor types analyzed. Especially in ovarian and esophageal carcinomas we found a clear tumor-selective overexpression of LOC121838.

The extracellular domains from LOC121838 according to the invention as a target structure of antibodies be used.

Example 18: Identification of LOC221103 as a diagnostic and therapeutic cancer target

The located in gene locus LOC221103 on chromosome 11 (11q12.3) gene with the transcript of SEQ ID NO: 69, the protein encodes with the SEQ ID NO: 70. The transmembrane protein does not show homology already known proteins.

According to the invention, after the establishment of a LOC221103-specific quantitative RT-PCR (primer pair SEQ ID NO: 71 and 72), the amount of gene-specific transcripts in healthy tissue and in carcinoma samples was investigated. LOC221103 is only expressed in the liver in the healthy tissues we examined and is otherwise undetectable ( 35 ). Surprisingly, LOC221103-specific transcripts are overexpressed in liver carcinomas. ( 36 ).

The extracellular domains from LOC221103 according to the invention as a target structure of antibodies be used.

Example 19: Identification of LOC338579 as a diagnostic and therapeutic cancer target

The located in gene locus LOC338579 on chromosome 10 (10q11.21) gene with the transcript of SEQ ID NO: 73, the protein encodes SEQ ID NO: 74. The transmembrane protein shows no homologies to already known proteins.

According to the invention, after the establishment of a LOC338579-specific quantitative RT-PCR (primer pair SEQ ID NO: 75 and 76), the amount of gene-specific transcripts in healthy tissue and in carcinoma samples was investigated. We found expression in healthy tissues exclusively in testis and weaker in the liver and thymus. Surprisingly, we found overexpression of LOC338579 in colon and liver carcinomas compared to healthy tissue ( 37 ).

The extracellular domains from LOC338579 according to the invention as a target structure of antibodies be used.

Example 20: Identification of LOC90342 as a diagnostic and therapeutic cancer target

The in gene locus LOC90342 on chromosome 2 (2q11.2) localized gene the transcript of SEQ ID NO: 77 encodes the protein having the SEQ ID NO: 78. The transmembrane protein contains a protein kinase C and various phospholipases conserved calcium-binding motif (CA1B).

According to the invention, after the establishment of a LOC90342-specific quantitative RT-PCR (primer pair SEQ ID NO: 79 and 80), the amount of gene-specific transcripts in healthy tissue and in carcinoma samples was investigated ( 38 ). We found LOC90342 only in a small number of healthy tissues, most of which are less toxic relevant ( 38 ). In contrast, we found LOC90342 specific transcripts in a variety of the tumor types analyzed. In particular in gastric, liver, pancreatic, prostate, ovarian and lung carcinomas, a partially clearly tumor-selective overexpression of LOC90342 was observed.

The Membrane protein has a single transmembrane domain (amino acids 707-726). The extracellular domain of LOC90342 can according to the invention as a target structure of therapeutic antibodies be used.

Example 21: Identification of LRFN1 as a diagnostic and therapeutic cancer target

LRFN1 (SEQ ID NO: 81) is a gene located on chromosome 19 (19q13.2) is. The gene codes for the protein of SEQ ID NO: 82 The protein contains a transmembrane domain and shows Homologies to the Myb DNA binding domain and to a C2-type immunoglobulin domain.

According to the invention, after the establishment of an LRFN1-specific quantitative RT-PCR (primer pair SEQ ID NO: 83 and 84), the amount of gene-specific transcripts in healthy tissue and in carcinoma samples was investigated. LRFN1 is very weakly expressed in most normal tissues except for activated PBMC and brain. ( 39 ). In contrast, we found increased evidence of LRFN1-specific transcripts in some of the tumor types analyzed. In particular in gastric, pancreatic, esophageal and breast carcinomas, we found a clear tumor-selective overexpression of LRFN1 in comparison to the associated normal tissues.

The protein contains a transmembrane domain (amino acids 448-470). The extracellular domains of LRFN1 can be used according to the invention as the target structure of therapeutic antibodies become.

Example 22: Identification of LOC285916 as a diagnostic and therapeutic cancer target

The located in gene locus LOC285916 on chromosome 7 (7p22.3) gene with the transcript of SEQ ID NO: 85, the protein encodes with the SEQ ID NO: 86. The transmembrane protein shows no homologies already known proteins.

According to the invention, after the establishment of a LOC285916-specific quantitative RT-PCR (primer pair SEQ ID NO: 87 and 88), the amount of gene-specific transcripts in healthy tissue and in carcinoma samples was investigated. LOC285916 is selectively expressed in testis in the healthy tissues we studied, but in all other tissues we were unable to detect the gene, or only to a limited extent ( 40A ). Surprisingly, we found LOC285916-specific transcripts in all tumor types tested. Particularly in breast, esophageal, renal, ENT and lung carcinomas a clear tumor-specific overexpression was detectable ( 40A and B).

The extracellular domains of LOC285916 (amino acids 42 to 93) according to the invention as a target structure of antibodies be used.

Example 23: Identification of MGC71744 as a diagnostic and therapeutic cancer target

The Gene MGC71744 with SEQ ID NO: 93 coded on chromosome 17 (17p13.2) the protein having SEQ ID NO: 94. The transmembrane protein does not show any Homologies to already known proteins.

According to the invention, after the establishment of a MGC71744-specific quantitative RT-PCR (primer pair SEQ ID NO: 95 and 96), the amount of gene-specific transcripts in healthy tissue and in carcinoma samples (pool of samples) was investigated ( 41 ). MGC71744 is hardly expressed in healthy tissue. Small amounts of specific transcripts were found only in the lung and spleen. Little or no detectable MGC71744-specific expression in all other healthy tissues analyzed ( 41A ). In contrast, we surprisingly found MGC71744-specific overexpression in some types of tumors, especially in renal carcinomas ( 41A and B) compared to healthy tissue.

Especially the extracellular domain of MGC71744 (N-terminus, amino acids 67-85) can be used according to the invention as a target structure of antibodies be used.

Example 24: Identification of LOC342982 as a diagnostic and therapeutic cancer target

The in gene locus LOC342982 on chromosome 19 (19p13.13) located gene with the transcript of SEQ ID NO: 97, the protein encodes SEQ ID NO: 98. The transmembrane protein shows homology to the carbohydrate-binding domain of the C-type the lectins.

According to the invention, after the establishment of a LOC342982-specific quantitative RT-PCR (primer pair SEQ ID NO: 99 and 100), the amount of gene-specific transcripts in healthy tissue and in carcinoma samples (pool of samples) was investigated. LOC342982-specific RNA is selectively expressed, in many normal tissues analyzed little or no expression was detectable ( 42 ). In contrast, almost all of the tumor classes tested show a partial tumor-specific overexpression. Mainly pancreatic, renal, lung and breast carcinomas show a very strong expression of the LOC342982-specific RNA ( 42 ).

Especially the extracellular domain of LOC342982 (amino acids 178-339) can according to the invention as a target structure of monoclonal antibodies be used.

Example 25: Identification of LOC343169 / OR6F1 as a Diagnostic and Therapeutic Cancer Target

The in gene locus LOC343169 on chromosome 1 (1q44) localized gene OR6F1 with the transcript of SEQ ID NO: 101, the protein encodes SEQ ID NO: 102. OR6F1 has multiple transmembrane domains and belongs to the olfactory family Receptors and thus the big one Family of G-protein coupled receptors.

According to the invention, after the establishment of a LOC343169 / OR6F1-specific quantitative RT-PCR (primer pair SEQ ID NO: 103 and 104), the amount of gene-specific transcripts in healthy tissue and in carcinoma samples (pool of samples) was investigated ( 43A ). LOC343169 / OR6F1 is expressed very selectively in healthy tissue, specific transcripts are detectable especially in testis and spleen. Little or no detectable LOC343169 / OR6F1-specific expression in all other healthy tissues analyzed ( 43A ). Surprisingly, however, LOC343169 / OR6F1-specific overexpression could be detected in some tumor types. Especially in breast, ovarian, kidney, prostate, pancreatic and hepatic carcinomas, a tumor-specific overexpression appears on by LOC343169 / OR6F1 ( 43A and B). Analysis of individual samples confirmed the overexpression in ovarian carcinomas.

LOC343169 / OR6F1 is a selectively expressed antigen that is evident in proliferating tissues reinforced is expressed. It is thus a selective overexpression in tumors too observe that is therapeutically usable. In particular, the extracellular domains can be used according to the invention as target structures of monoclonal antibodies be used.

Example 26: Identification of LOC340204 as a diagnostic and therapeutic cancer target

The located in gene locus LOC340204 on chromosome 6 (6p21.31) gene with the transcript of SEQ ID NO: 105, the protein encodes with the SEQ ID NO: 106. The protein has a transmembrane domain. About it shows the protein has strong homology to a "colipase" domain. Colipase is believed to function as a cofactor for pancreatic lipase. An alternative transcript of the gene locus is represented by SEQ ID NO: 279 and the gene product SEQ ID NO: 280 and could both an independent one Transcript as well as a splice variant SEQ ID NO: 105.

According to the invention, after the establishment of a LOC340204-specific quantitative RT-PCR (primer pair SEQ ID NO: 107 and 108), the amount of gene-specific transcripts in healthy tissue and in carcinoma samples was investigated. LOC340204 is selectively expressed in healthy tissues and strongly overexpressed in some tumors. Especially in gastric, pancreatic, ovarian, lung and esophageal carcinomas ( 44A and B) a clear overexpression in tumor samples could be detected compared to different normal tissues.

The extracellular domains from LOC340204 according to the invention as a target structure of monoclonal antibodies be used.

Example 27: Identification of LOC340067 as a diagnostic and therapeutic cancer target

The located in gene locus LOC340067 on chromosome 5 (5q22.3) gene with the transcript of SEQ ID NO: 109, the protein codes with the SEQ ID NO: 110. The transmembrane protein does not show homology other protein domains.

According to the invention, after the establishment of a quantitative RT-PCR specific for the LOC340067 (primer pair SEQ ID NO: 111 and 112), the amount of gene-specific transcripts in healthy tissue and in carcinoma samples was investigated. LOC340067 is selectively expressed in healthy tissues and strongly overexpressed in some tumors ( 45 ). Especially in pancreatic, breast, liver, ovarian, lung and renal carcinomas a clear overexpression in tumor samples could be detected compared to different healthy tissues.

The extracellular domain of LOC340067 can according to the invention as a target structure of monoclonal antibodies be used.

Example 28: Identification of LOC342780 as a diagnostic and therapeutic cancer target

The located in gene locus LOC342780 on chromosome 18 (18q21.32) gene with the transcript of SEQ ID NO: 309, the protein encodes SEQ ID NO: 310. The transmembrane protein contains an acyltransferase domain, the in many previously uncharacterized proteins from C. elegans occurs.

According to the invention, after the establishment of a LOC342780-specific quantitative RT-PCR (primer pair SEQ ID NO: 311 and 312), the amount of gene-specific transcripts in healthy tissue and in carcinoma samples (pool of samples, the number is shown in the figure) was investigated. LOC342780 is very selectively expressed in healthy tissue, specific transcripts are detectable mainly in the prostate, stomach, testis, lung and mammary gland ( 46 ). In contrast, LOC342780-specific expression was surprisingly detected in all tumor types analyzed. Particularly in breast, ovarian, kidney and liver carcinomas, a tumor-specific overexpression of LOC342780 ( 46 ).

LOC342780 is a selectively expressed antigen that is apparently proliferating Reinforced tissues is expressed. It is thus a selective overexpression in tumors too observe that is therapeutically usable. The extracellular localized amino acids 76-89, 316-345, 399-493 and 650-665 (based on SEQ ID NO: 310) can be used according to the invention as target structures of monoclonal antibodies be used.

Example 29: Identification of LOC339511 as a diagnostic and therapeutic cancer target

The Sequence according to SEQ ID NO: 113 is a gene located on chromosome 1 (1q23.1). The Gene encodes the protein according to SEQ ID NO: 114. The transmembrane protein shows homology to the group of olfactory 7-transmembrane receptors.

According to the invention was after Etablie a quantity of gene-specific transcripts in healthy tissue and in carcinoma samples was investigated for a quantitative RT-PCR (primer pair SEQ ID NO: 115 and 116) specific for LOC339511. LOC339511 is selectively expressed in the liver in healthy tissues ( 47A ). In the carcinoma samples, LOC339511-specific transcripts were identified in liver tumors, and weak expression was detectable in colon, breast and lung carcinomas. When comparing the liver-specific expression in tumor and healthy tissues increased expression was detected in some tumor samples ( 47B ).

The extracellular domains of SEQ ID NO: 113 According to the invention as target structures of monoclonal antibodies be used. In particular, the extracellular localized amino acid residues 1-23, 82-100, 167-175, 226-236 are therefore particularly suitable for the preparation of monoclonal antibodies.

Example 30: Identification the sequence C14orf37 (SEQ ID NO: 125/126) as a diagnostic and therapeutic cancer target

C14orf37 (SEQ ID NO: 125) is located on chromosome 14 (14q22.3) A gene encoding the gene product having SEQ ID NO: 126. The transmembrane protein shows no homologies to already known proteins.

According to the invention, after the establishment of a C14orf37-specific quantitative RT-PCR (primer pair SEQ ID NO: 127 and 128), the amount of gene-specific transcripts in healthy tissue and carcinoma samples was investigated. The C14orf37 is expressed in different healthy tissues and most strongly in the testis ( 48 ). Especially in renal carcinomas a clear overexpression could be detected compared to different healthy tissues.

The extracellular domain SEQ ID NO 126 can according to the invention as a target structure of monoclonal antibodies be used.

Example 31: Identification of ATP1A4 as a diagnostic and therapeutic cancer target

The Gene ATP1A4 (SEQ ID NO: 129) is located on chromosome 1 (1q21-23). The gene codes for a protein according to SEQ ID NO: 130. ATP1A4 is an integral transmembrane protein with eight Transmembrane domains, which is located in the plasma membrane. ATP1A4 is part of a Protein complex, wherein the catalytic part of the sodium / potassium ATPase N-terminal (Woo et al., J. 2000. Biol. Chem. 275, 20693-99). ATP1A4 shows strong homologies to many other members of the cation ATPase family.

According to the invention, after the establishment of an ATP1A4-specific quantitative RT-PCR (primer pair SEQ ID NO: 131 and 132), the amount of gene-specific transcripts in healthy tissue and in carcinoma samples was investigated. ATP1A4 is selectively expressed in healthy tissues, especially in the testis ( 49A ). In some tumor samples, a strong over-expression of ATP1A4 was detected in comparison to the respective healthy tissue. Especially in pancreatic, breast, liver and renal carcinomas ( 49A and B) a clear overexpression in tumor samples compared to healthy tissues could be detected, overall very high expression was in pancreatic and breast carcinomas.

The extracellular domains from ATP1A4 according to the invention as a target structure of monoclonal antibodies be used. The following amino acid residues with respect to SEQ ID NO 130 are extracellular localized: amino acid residues 129-137, 321-329, 816-857, and 977-990.

Example 32: Identification SEQ ID NO: 133 to 264 as a diagnostic and therapeutic Cancer Targets

at SEQ ID NO: 133-266 is a total of 33 genes each including nucleic acid sequence, Amino acid sequence, "sense" PCR primer and "antisense" PCR primer for specific RT-PCR Reactions. All proteins have one or multiple transmembrane domains, via homologies to protein domains is little known.

According to the invention were for this Genes in specific quantitative RT-PCR reactions the amount of respective gene-specific transcripts in healthy tissue and in Carcinoma samples examined. For all genes could be compared to the respective one in tumor samples healthy tissue, partly strong overexpression be detected.

The Group of these genes can be used therapeutically and diagnostically. The extracellular domains can according to the invention as a target structure of antibodies be used.

It follows a sequence listing according to WIPO St. 25. This can from the official publication platform downloaded from the DPMA.

Claims (114)

Pharmaceutical composition, comprising send an agent that inhibits the expression or activity of a tumor-associated antigen, wherein the tumor-associated antigen has a sequence encoded by a nucleic acid selected from the group consisting of: (a) a nucleic acid, the a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65 , 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165 , 169, 173, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263 , 267, 269, 271, 273, 275, 277, 279, 309 of the Sequence Listing, a part or derivative thereof, (b) a nucleic acid which hybridizes under stringent conditions with the nucleic acid under (a), (c) a nucleic acid, which is degenerate with respect to the nucleic acid under (a) or (b), and (d) a nucleic acid, which is complementary to the nucleic acid of (a), (b) or (c). A pharmaceutical composition comprising an agent with antitumor activity, that is selective for Cells that cause expression or abnormal expression of a tumor Antigen, wherein the tumor-associated antigen is a sequence which encodes a nucleic acid is selected from the group is consisting of (a) a nucleic acid containing a nucleic acid sequence which is selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 269, 271, 273, 275, 277, 279, 309 of the Sequence Listing, a part or Derivative thereof, (b) a nucleic acid that is under stringent Conditions with the nucleic acid hybridized under (a), (c) a nucleic acid which is in relation to the nucleic acid (a) or (b) is degenerate, and (d) a nucleic acid which to the nucleic acid under (a), (b) or (c) is complementary. Pharmaceutical composition according to claim 2, wherein the agent is the induction of cell death, the reduction of cell growth, a damage the cell membrane or secretion of cytokines causes. A pharmaceutical composition according to claim 1 or 2, wherein the agent is an antisense nucleic acid which selectively binds with the Nucleic acid hybridizes, the for the tumor-associated Encoded antigen. A pharmaceutical composition according to claim 1 or 2, wherein the agent is an antibody, which binds selectively to the tumor-associated antigen. Pharmaceutical composition according to claim 2, wherein the agent is a complement-activating antibody, which binds selectively to the tumor-associated antigen. A pharmaceutical composition comprising a means which when administered selectively the amount of complexes between an HLA molecule and a tumor-associated antigen or part thereof, wherein the tumor-associated antigen has a sequence derived from a nucleic acid which is selected from the group consisting of: (A) a nucleic acid, the one nucleic acid sequence which is selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 269, 271, 273, 275, 277, 279, 309 of the Sequence Listing, a part or Derivative thereof, (b) a nucleic acid that is under stringent Conditions with the nucleic acid hybridized under (a), (c) a nucleic acid which is in relation to the nucleic acid (a) or (b) is degenerate, and (d) a nucleic acid which to the nucleic acid under (a), (b) or (c) is complementary. Pharmaceutical composition according to claim 7, wherein the agent comprises one or more components consisting of the group are selected consisting of: (i) the tumor-associated antigen or a Part of it, (ii) a nucleic acid encoding the tumor-associated antigen or encoded part of it, (iii) a host cell, the expressing the tumor-associated antigen or a part thereof, and (iv) isolated complexes between the tumor-associated Antigen or a part thereof and an HLA molecule. A pharmaceutical composition according to claim 1, 2 or 7 wherein said agent comprises a plurality of agents each selectively inhibiting the expression or activity of various tumor-associated antigens, each being selective for cells expressing different tumor-associated antigens or the amount of complexes between HLA molecules and various tumor-associated antigens or parts thereof, wherein at least one of the tumor-associated antigens has a sequence which is encoded by a nucleic acid selected from the group consisting of: (a) a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13 , 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113 , 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211 , 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 269, 271, 273, 275, 277, 279, 309 of the Sequence Listing, a part or derivative thereof (b) a nucleic acid which hybridizes under stringent conditions with the nucleic acid under (a), (c) a nucleic acid which is degenerate with respect to the nucleic acid under (a) or (b), and (d) a nucleic acid, which is complementary to the nucleic acid of (a), (b) or (c). Pharmaceutical composition comprising a or more ingredients selected from the group consisting of: (i) a tumor-associated antigen or a Part of it, (ii) a nucleic acid encoding a tumor-associated antigen or encoded part of it, (iii) an antibody which binds to a tumor-associated antigen or a part thereof, (Iv) an antisense nucleic acid, which hybridizes specifically with a nucleic acid encoding a tumor-associated antigen, (V) a host cell containing a tumor-associated antigen or a Part of it expressed, and (vi) isolated complexes between a tumor-associated antigen or a part thereof and an HLA molecule, in which the tumor-associated antigen has a sequence derived from a nucleic acid which is selected from the group consisting of: (A) a nucleic acid, the one nucleic acid sequence which is selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 269, 271, 273, 275, 277, 279, 309 of the Sequence Listing, a part or Derivative thereof, (b) a nucleic acid that is under stringent Conditions with the nucleic acid hybridized under (a), (c) a nucleic acid which is in relation to the nucleic acid (a) or (b) is degenerate, and (d) a nucleic acid which to the nucleic acid under (a), (b) or (c) is complementary. Pharmaceutical composition according to claim 8 or 10, wherein the nucleic acid under (ii) is present in an expression vector. Pharmaceutical composition according to claim 8 or 10, wherein the nucleic acid under (ii) is operably linked to a promoter. Pharmaceutical composition according to claim 8 or 10, wherein the host cell is the tumor-associated antigen or the part of it secreted. Pharmaceutical composition according to claim 8 or 10, wherein the host cell additionally expresses an HLA molecule, which binds to the tumor-associated antigen or part thereof. Pharmaceutical composition according to claim 14, wherein the host cell is the HLA molecule and / or the tumor-associated Antigen or the part thereof expressed recombinantly. Pharmaceutical composition according to claim 14, wherein the host cell endogenously expresses the HLA molecule. Pharmaceutical composition according to claim 8, 10, 14 or 16, wherein the host cell is an antigen-presenting Cell is. Pharmaceutical composition according to claim 17, being the antigen presenting Cell is a dendritic cell, a monocyte or macrophage. Pharmaceutical composition according to one of claims 8, 10 and 13-18, wherein the host cell is non-proliferative. Pharmaceutical composition according to claim 5 or 10, wherein the antibody a monoclonal antibody is. Pharmaceutical composition according to claim 5 or 10, wherein the antibody a chimera or humanized antibody is. Pharmaceutical composition according to claim 5 or 10, wherein the antibody a fragment of a natural one antibody is. Pharmaceutical composition according to claim 5 or 10, wherein the antibody coupled with a therapeutic or diagnostic agent. Pharmaceutical composition according to claim 4 or 10, wherein the antisense nucleic acid is a Sequence of 6-50 contiguous nucleotides from the nucleic acid, the for encodes the tumor-associated antigen. A pharmaceutical composition according to any one of claims 8 and 10-13, provided by the pharmaceutical composition Tumor-associated antigen or part of it to MHC molecules on the surface of cells that bind an abnormal amount of the tumor Antigen or part of it. Pharmaceutical composition according to claim 25, wherein the binding causes a cytolytic reaction and / or a cytokine secretion induced Pharmaceutical composition according to any one of claims 1-26, further comprising a pharmaceutically acceptable carrier and / or adjuvant. A pharmaceutical composition according to claim 27, wherein the adjuvant is saponin, GM-CSF, a CpG nucleotide, cytokine or Chemokine is. Pharmaceutical composition according to any one of claims 1-28, which can be used to treat a disease that itself by the expression or abnormal expression of a tumor-associated Antigens distinguished. Pharmaceutical composition according to claim 29, where the disease is cancer. Pharmaceutical composition according to claim 29, the disease being a colon, rectal, kidney, adrenal, Breast, prostate, uterus, Ovarian, endometrial, esophageal, Blood, liver, pancreas, skin, brain or lung cancer, a lymphoma or neuroblastoma, a lung, breast, prostate, colon tumor, renal cell, Cervical, colon or breast carcinoma or metastases of the above Cancers or tumors. Pharmaceutical composition according to any one of claims 1-31, wherein the tumor-associated antigen comprises an amino acid sequence comprising the group selected is, consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 270, 272, 274, 276, 278, 280 to 308, 310 of the Sequence Listing, a part or derivative thereof. Procedure for diagnosing a disease that is by the expression or abnormal expression of a tumor-associated Antigen characterized, comprising (i) the detection of a nucleic acid which for the Tumor-associated antigen encoded, or part thereof, and / or (Ii) the detection of the tumor-associated antigen or a part thereof, and or (iii) the detection of an antibody to the tumor-associated Antigen or a part thereof and / or (iv) proof of cytotoxic or helper T lymphocytes responsible for the tumor-associated antigen or part of which are specific in a biological isolated from a patient Sample, wherein the tumor-associated antigen has a sequence, that of a nucleic acid which is selected from the group consisting of: (A) a nucleic acid, the one nucleic acid sequence which is selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 269, 271, 273, 275, 277, 279, 309 of the Sequence Listing, a part or Derivative thereof, (b) a nucleic acid that is under stringent Conditions with the nucleic acid hybridized under (a), (c) a nucleic acid which is in relation to the nucleic acid (a) or (b) is degenerate, and (d) a nucleic acid which to the nucleic acid under (a), (b) or (c) is complementary. The method of claim 33, wherein the detection (I) contacting the biological sample with an agent that is specific to the nucleic acid, the for the tumor-associated antigen encodes, or the portion thereof, to the Tumor-associated antigen or the part thereof, to the antibody or binds to the cytotoxic or helper T lymphocytes, and (Ii) the detection of complex formation between the agent and the nucleic acid or the part thereof, the tumor-associated antigen or the part thereof, the antibody or the cytotoxic or helper T lymphocytes. The method of claim 33 or 34, wherein the detection with evidence in a comparable normal biological sample is compared. The method of any of claims 33-35, wherein the disease is characterized by the expression or abnormal expression of a plurality of different tumor-associated antigens, and the detection comprises detection of a plurality of nucleic acids encoding the plurality of different tumor-associated antigens or portions thereof; the proof of several different tumor asso cited antigens or portions thereof, the detection of multiple antibodies that bind to the several different tumor-associated antigens or portions thereof, or the detection of multiple cytotoxic or helper T lymphocytes specific for the several different tumor-associated antigens , includes. The method of any of claims 33-36, wherein the detection the nucleic acid or the part thereof with a polynucleotide probe, the specifically with the nucleic acid or the part thereof hybridized. The method of claim 37, wherein the polynucleotide probe a sequence of 6-50 contiguous Nucleotides from the nucleic acid, the for the Encodes tumor-associated antigen. The method of any of claims 33-36, wherein the detection the nucleic acid or the part thereof by selective amplification of the nucleic acid or the part of it takes place. A method according to any one of claims 33-36, wherein the to be detected Tumor-associated Antigen or part thereof in a complex with an MHC molecule. The method of claim 40, wherein the MHC molecule is an HLA molecule. A method according to any one of claims 33-36 and 40-41, wherein the detection of the tumor-associated antigen or part thereof with an antibody carried out specifically to the tumor-associated antigen or the Part of it binds. The method of any of claims 33-36, wherein the detection of the antibody with a protein or peptide that binds specifically to the antibody. Method for determining the regression, the course or the onset of a disease characterized by expression or abnormal expression of a tumor-associated antigen, comprising The supervision a sample from a patient having the disease or is suspected of having the disease in relation to one or more parameters selected from the group out: (i) the amount of nucleic acid responsible for the tumor-associated antigen encoded, or part of it, (ii) the amount of tumor-associated Antigen or part of it, (iii) the amount of antibodies that bind to the tumor-associated antigen or a part thereof, and (Iv) the amount of cytolytic or cytokine-releasing T cells responsible for a Complex between the tumor-associated antigen or a part thereof and an MHC molecule specific with the tumor-associated antigen having a sequence, that of a nucleic acid which is selected from the group consisting of: (A) a nucleic acid, the one nucleic acid sequence which is selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 269, 271, 273, 275, 277, 279, 309 of the Sequence Listing, a part or Derivative thereof, (b) a nucleic acid that is under stringent Conditions with the nucleic acid hybridized under (a), (c) a nucleic acid which is in relation to the nucleic acid (a) or (b) is degenerate, and (d) a nucleic acid which to the nucleic acid under (a), (b) or (c) is complementary. The method of claim 44, wherein the method the determination of the parameter (s) at a first time in a first sample and at a second time in another Sample includes and by comparing the two samples of the course the disease is determined. The method of claim 44 or 45, wherein the disease by the expression or abnormal expression of several different Tumor-associated antigens and surveillance monitoring (I) the amount of several nucleic acids, the for the several different tumor-associated Encode antigens, or parts of them, (ii) the amount of several different tumor-associated antigens or parts from that, (iii) the amount of several antibodies that belong to the several different Tumor-associated antigens or bind to parts thereof, and / or (Iv) the amount of multiple cytolytic or cytokine secretagogues T cells used for Complexes between the several different tumor-associated Antigens or parts thereof and MHC molecules. The method of any one of claims 44-46, wherein the monitoring the amount of nucleic acid or the part thereof is carried out with a polynucleotide probe specific with the nucleic acid or the part thereof hybridized. The method of claim 47, wherein the polynucleotide probe a sequence of 6-50 contiguous Nucleotides from the nucleic acid, the for the Encodes tumor-associated antigen. The method of any one of claims 44-46, wherein the monitoring the amount of nucleic acid or the part thereof by selective amplification of the nucleic acid or the part of it takes place. The method of any one of claims 44-46, wherein the monitoring the amount of the tumor-associated antigen or part thereof an antibody carried out specifically to the tumor-associated antigen or the Part of it binds. The method of any one of claims 44-46, wherein the monitoring the amount of antibodies with a protein or peptide that binds specifically to the antibody. The method of any one of claims 44-46, wherein the monitoring the amount of cytolytic or cytokine secretory T cells having a Cell takes place, which is the complex between the tumor-associated antigen or the part thereof and an MHC molecule. A method according to any of claims 37-38, 42-43, 47-48 and 50-52, wherein the polynucleotide probe, the antibody, the protein or peptide or the cell is detectably marked. The method of claim 53, wherein the detectable Marker is a radioactive marker or an enzyme label. The method of any of claims 33-54, wherein the sample body fluid and / or body tissue includes. Method of treating a disease that has become by the expression or abnormal expression of a tumor-associated Antigens comprising the administration of a pharmaceutical A composition according to any one of claims 1-32, wherein the tumor-associated Antigen has a sequence that encodes a nucleic acid is selected from the group is composed of: (a) a nucleic acid containing a nucleic acid sequence which is selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 269, 271, 273, 275, 277, 279, 309 of the Sequence Listing, a part or Derivative thereof, (b) a nucleic acid that is under stringent Conditions with the nucleic acid hybridized under (a), (c) a nucleic acid which is in relation to the nucleic acid (a) or (b) is degenerate, and (d) a nucleic acid which to the nucleic acid under (a), (b) or (c) is complementary. Method of treatment, diagnosis or monitoring a disorder characterized by expression or abnormal expression a tumor-associated antigen, comprising the administration an antibody, which binds to the tumor-associated antigen or a part thereof and coupled with a therapeutic or diagnostic agent, wherein the tumor-associated antigen has a sequence derived from a nucleic acid which is selected from the group consisting of: (A) a nucleic acid, the one nucleic acid sequence which is selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 269, 271, 273, 275, 277, 279, 309 of the Sequence Listing, a part or Derivative thereof, (b) a nucleic acid that is under stringent Conditions with the nucleic acid hybridized under (a), (c) a nucleic acid which is in relation to the nucleic acid (a) or (b) is degenerate, and (d) a nucleic acid which to the nucleic acid under (a), (b) or (c) is complementary. A method according to claim 42, 50 or 57, wherein the antibody a monoclonal antibody is. A method according to claim 42, 50 or 57, wherein the antibody a chimera or humanized antibody is. A method according to claim 42, 50 or 57, wherein the antibody a fragment of a natural one antibody is. A method of treating a patient with a disease characterized by the expression or abnormal expression of a tumor-associated antigen comprising: (i) removing a sample from the patient with immunoreactive cells, (ii) contacting the sample with a host cell expressing the tumor-associated antigen or portion thereof, under conditions which favor production of cytolytic or cytokine-releasing T cells against the tumor-associated antigen or portion thereof, and (iii) introducing the cytolytic or cytokine antigens. Injecting T cells into the patient in an amount capable of lysing cells expressing the tumor-associated antigen or a portion thereof, wherein the tumor-associated antigen A sequence encoded by a nucleic acid selected from the group consisting of: (a) a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13 , 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113 , 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211 , 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 269, 271, 273, 275, 277, 279, 309 of the Sequence Listing, a part or derivative thereof (b) a nucleic acid which hybridizes under stringent conditions with the nucleic acid under (a), (c) a nucleic acid which is degenerate with respect to the nucleic acid under (a) or (b), and (d) a nucleic acid, which is complementary to the nucleic acid of (a), (b) or (c). The method of claim 61, wherein the host cell an HLA molecule expressed recombinantly to the tumor-associated antigen or binds a part of it. The method of claim 62, wherein the host cell an HLA molecule expressed endogenously to the tumor-associated antigen or a Part of it binds. Method of treating a patient with a patient Disease resulting from expression or abnormal expression of a tumor-associated antigen, comprising: (I) the identification of a nucleic acid that is expressed by cells is associated with the disease, wherein the nucleic acid from the group selected is composed of: (a) a nucleic acid containing a nucleic acid sequence which is selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 269, 271, 273, 275, 277, 279, 309 of the Sequence Listing, a part or Derivative thereof, (b) a nucleic acid that is under stringent Conditions with the nucleic acid hybridized under (a), (c) a nucleic acid which is in relation to the nucleic acid (a) or (b) is degenerate, and (d) a nucleic acid which to the nucleic acid is complementary under (a), (b) or (c), (ii) transfection a host cell with the nucleic acid or a part thereof, (Iii) culturing the transfected host cell for expression of the nucleic acid, and (Iv) introducing the host cells or an extract thereof into the Patients in an amount that is appropriate to the immune response the cells of the patient associated with the disease to increase. The method of claim 64, further comprising Identification of an MHC molecule containing the Presenting tumor-associated antigen or part thereof, wherein the host cell expresses the identified MHC molecule and presenting the tumor-associated antigen or part thereof. The method of claim 64 or 65, wherein the immune reaction a B-cell reaction or a T-cell reaction. The method of claim 66, wherein the immune response is a T-cell reaction comprising cytolytic production or cytokine-releasing T cells specific for the host cells are the tumor-associated antigen or part present it or specific to Cells of the patient are the tumor-associated antigen or express a part of it. The method of any of claims 61-67, wherein the host cells are non-proliferative. A method of treating a disorder characterized by the expression or abnormal expression of a tumor-associated antigen, comprising: (i) identifying cells from the patient that express abnormal levels of the tumor-associated antigen, (ii) isolating a patient (Iii) culturing the cells, and (iv) introducing the cells into the patient in an amount effective to induce an immune response to the cells, the tumor-associated antigen having a sequence derived from a nucleic acid selected from the group consisting of: (a) a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 175, 179, 183, 187, 191, 195, 199, 203, 2 07, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 269, 271, 273, 275, 277, 279, 309 of the Sequence Listing, a part or derivative thereof, (b) a nucleic acid which hybridizes under stringent conditions with the nucleic acid under (a), (c) a nucleic acid which is degenerate with respect to the nucleic acid under (a) or (b), and (d) a nucleic acid which is linked to the nucleic acid (a), (b) or (c) is complementary. The method of any of claims 33-69, wherein the disease Cancer is. Method of inhibiting the development of cancer in a patient comprising the administration of an effective Amount of a pharmaceutical composition according to any one of claims 1-32. The method of any of claims 33-71, wherein the tumor-associated Antigen an amino acid sequence which is selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 270, 272, 274, 276, 278, 280 to 308, 310 of the Sequence Listing, a part or Derivative thereof. Nucleic acid, selected from the group is composed of: (a) a nucleic acid containing a nucleic acid sequence which is selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 269, 271, 273, 275, 277, 279, 309 of the Sequence Listing, a part or Derivative thereof, (b) a nucleic acid that is under stringent Conditions with the nucleic acid hybridized under (a), (c) a nucleic acid which is in relation to the nucleic acid (a) or (b) is degenerate, and (d) a nucleic acid which to the nucleic acid under (a), (b) or (c) is complementary. Nucleic acid, the for encodes a protein or polypeptide having an amino acid sequence which is selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 270, 272, 274, 276, 278, 280 to 308, 310 of the Sequence Listing, a Part or derivative thereof. Recombinant DNA or RNA molecule containing a nucleic acid according to claim 73 or 74. The recombinant DNA molecule of claim 75, wherein said recombinant DNA molecule is a vector. The recombinant DNA molecule of claim 76, wherein said Vector is a viral vector or bacteriophage. Recombinant DNA molecule according to any one of claims 75-77, which further comprises expression control sequences expressing expression the nucleic acid Taxes. The recombinant DNA molecule of claim 78, wherein said Expression control sequences are homo- or heterologous to the nucleic acid. A host cell comprising a nucleic acid according to claim 73 or 74 or a recombinant DNA molecule according to one of the claims 75-79. The host cell of claim 80, further comprising a nucleic acid, the for encodes an HLA molecule. Protein or polypeptide derived from a nucleic acid Claim 73 is encoded. Protein or polypeptide having an amino acid sequence which is selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 270, 272, 274, 276, 278, 280 to 308, 310 of the Sequence Listing, a part or derivative thereof. Immunogenic fragment of the protein or polypeptide according to claim 82 or 83. Fragment of the protein or polypeptide according to claim 82 or 83 attached to a human HLA receptor or human Antibody binds. An agent that specifically binds to a protein or polypeptide or to a portion thereof, wherein the protein or polypeptide is encoded by a nucleic acid selected from the group consisting of: (a) a nucleic acid comprising a nucleic acid sequence derived from the A group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 269, 271, 273, 275, 277, 279, 309 of the Sequence Listing, a part or derivative thereof, (b) a nucleic acid which hybridizes under stringent conditions with the nucleic acid under (a), (c) a nucleic acid which is degenerate with respect to the nucleic acid under (a) or (b), and (d) a nucleic acid complementary to the nucleic acid of (a), (b) or (c). The agent of claim 86, wherein the protein or Polypeptide an amino acid sequence which is selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 270, 272, 274, 276, 278, 280 to 308, 310 of the Sequence Listing, a Part or derivative thereof. Means according to claim 86 or 87, wherein the means an antibody is. The agent of claim 88, wherein the antibody comprises monoclonal, chimeric or humanized antibody or a fragment of an antibody is. Antibody, the selective to a complex of: (i) a protein or polypeptide or part of it and (ii) binds to an MHC molecule, to which the protein or polypeptide or part thereof binds, wherein the antibody does not bind to (i) or (ii) alone and the protein or polypeptide from a nucleic acid which is selected from the group consisting of: (A) a nucleic acid, the one nucleic acid sequence which is selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 269, 271, 273, 275, 277, 279, 309 of the Sequence Listing, a part or Derivative thereof, (b) a nucleic acid that is under stringent Conditions with the nucleic acid hybridized under (a), (c) a nucleic acid which is in relation to the nucleic acid (a) or (b) is degenerate, and (d) a nucleic acid which to the nucleic acid under (a), (b) or (c) is complementary. antibody according to claim 90, wherein the protein or polypeptide has an amino acid sequence which is selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 270, 272, 274, 276, 278, 280 to 308, 310 of the Sequence Listing, a Part or derivative thereof. antibody according to claim 90 or 91, wherein the antibody is a monoclonal, chimeric or humanized antibody or a fragment of an antibody is. A conjugate between an agent according to any of claims 86-89 or an antibody according to one of the claims 90-92 and a therapeutic or diagnostic agent. A conjugate according to claim 93, wherein the therapeutic or diagnostic agent is a toxin. Kit for detection of expression or abnormal Expression of a tumor-associated Antigens, comprising means for detection (i) the nucleic acid which for the Encodes tumor-associated antigen, or part of it, (Ii) the tumor-associated antigen or a part thereof, (Iii) of antibodies, which bind to the tumor-associated antigen or a part thereof, and or (iv) T cells responsible for a complex between the Tumor-associated antigen or a part thereof and an MHC molecule specific with the tumor-associated antigen having a sequence, that of a nucleic acid which is selected from the group consisting of: (A) a nucleic acid, the one nucleic acid sequence which is selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 269, 271, 273, 275, 277, 279, 309 of the Sequence Listing, a part or Derivative thereof, (b) a nucleic acid that is under stringent Conditions with the nucleic acid hybridized under (a), (c) a nucleic acid which is in relation to the nucleic acid (a) or (b) is degenerate, and (d) a nucleic acid which to the nucleic acid under (a), (b) or (c) is complementary. The kit of claim 95, wherein the means for detecting the nucleic acid responsible for the tumor asso ziert antigen, or a part thereof nucleic acid molecules for the selective amplification of the nucleic acid. The kit of claim 96, wherein the nucleic acid molecules for the selective Amplification of the nucleic acid a sequence of 6-50 contiguous Nucleotides from the nucleic acid, the for which encodes the tumor-associated antigen. Recombinant DNA molecule comprising a promoter region, that of a nucleic acid which comprises a nucleic acid sequence comprising the group selected is, consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 269, 271, 273, 275, 277, 279, 309 of the Sequence Listing. A pharmaceutical composition comprising a means that the expression or activity of the Tumor antigens according to SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 270, 272, 274, 276, 278, 280 to 308, 310 of the Sequence Listing. Antibody, the extracellular Areas of proteins comprising a sequence according to SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 270, 272, 274, 276, 278, 280 to 308, 310 of the Sequence Listing. Pharmaceutical composition according to claim 99, wherein the agent is an antisense nucleic acid which selectively interacts with the nucleic acid hybridized for the tumor antigens encoded. Pharmaceutical composition according to claim 101, wherein the antisense nucleic acid is a Sequence of 6-50 contiguous Nucleotides from the nucleic acids, the for which encode tumor antigens. Pharmaceutical composition according to claim 99, wherein the agent is RNA interference (RNAi). A pharmaceutical composition according to claim 103, wherein the RNAi contains a so-called. short hairpin structure (shRNA). Pharmaceutical composition according to claim 104, being shRNA by transcription after transfection with expression vectors arise. Pharmaceutical composition according to claim 104, wherein shRNA is produced by transcription of retroviruses Pharmaceutical composition according to claim 104, wherein shRNA is mediated by lentiviral systems. Pharmaceutical composition according to claim 99, wherein the agent is a small chemical molecule. A pharmaceutical composition according to claim 108, wherein the small chemical molecules bind to the tumor antigens A pharmaceutical composition according to claim 109, taking the small chemical molecules to the extracellular areas of proteins comprising a sequence according to SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 270, 272, 274, 276, 278, 280 to 308, 310 of the Sequence Listing. A method of treatment diagnosing or monitoring a metastatic tumor characterized by the expression or abnormal expression of at least one tumor antigen comprising administering an antibody that binds to at least one of the tumor antigens or a portion thereof and is coupled to a therapeutic or diagnostic agent, wherein the at least one tumor antigen has a sequence encoded by a nucleic acid consisting of: (a) a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 269, 271, 273, 275, 277, 279, 309 of the Sequence Listing, a part or derivative thereof, (b) a nucleic acid which hybridizes under stringent conditions with the nucleic acid under (a), (c) a nucleic acid which is degenerate with respect to the nucleic acid under (a) or (b) and (d) a nucleic acid which belongs to the nucleic acid in (a), (b) or (c) is complementary. The method of claim 111, wherein the antibody comprises monoclonal antibody is. The method of claim 111, wherein the antibody comprises chimeric or humanized antibody. The method of claim 111, wherein the antibody comprises Fragment of a natural Antibody is.